Journal: International Journal of Molecular Sciences

Article Title: TGF-β Inhibition Through Combinatory Strategies Suppresses Proliferation and Invasiveness in Malignant Pleural Mesothelioma

doi: 10.3390/ijms27052157

Figure Lengend Snippet: Efficacy of TGF-β silencing and its effect on cell viability and proliferation . ( A ) Percentage of TGF-β inhibition across all MPM cell lines (MSTO-211H, MM404, MM421, MM432) following transfection with TGF-β siRNA (60 nM). Data are presented as means ± SD ( n = 3). **** p < 0.0001, *** p < 0.001: siTGF-β vs. respective Ctrl. ( B ) Evaluation of the cell proliferation marker PCNA by real-time PCR 48 h after transfection. Data are presented as means ± SD ( n = 3). *** p < 0.001, ** p < 0.01: siTGF-β vs. respective Ctrl. ( C ) Evaluation of cell viability via MTT assay 48 h after transfection with TGF-β siRNA (60 nM). Measurements were performed in triplicate. Data are expressed as percentage of viable cells relative to control and presented as means ± SD ( n = 3). *** p < 0.001, ** p < 0.01: siTGF-β vs. respective Ctrl.

Article Snippet: Experiments were performed on the commercially available MPM cell line MSTO-211H (human biphasic mesothelioma cells), purchased from American Type Culture Collection (ATCC; Manassas, VA, USA), and primary MPM patient-derived cell lines MM404 (human epithelioid histotype), MM421 (human biphasic histotype) and MM432 (human sarcomatoid histotype) from Biological Bank of Mesothelioma (SS.

Techniques: Inhibition, Transfection, Marker, Real-time Polymerase Chain Reaction, MTT Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: TGF-β Inhibition Through Combinatory Strategies Suppresses Proliferation and Invasiveness in Malignant Pleural Mesothelioma

doi: 10.3390/ijms27052157

Figure Lengend Snippet: Effect of TGF-β silencing on MPM cell migration and cell cycle. ( A ) Scratch assay evaluated on MSTO-211H cells after 48 h without (-) or with 60 nM TGF-β siRNA. Cells were analyzed by microscopy at the time of scratching (T0) and after 24 h (T1). Relative migration was calculated by setting the percentage migration of control cells at T1 equal to 100% and comparing the percentage migration of cells after transfection with siTGF-β to this value. Data are presented as means ± SD. **** p < 0.0001: cells transfected with siTGF-β vs. Ctrl cells. ( B ) Cell cycle analysis using flow cytometry after staining with propidium iodide (PI). ( C ) Distribution of cells in sub-G1, G1/G0, S, and G2/M phases of the cell cycle under different experimental conditions. Data are presented as means ± SD ( n = 3). **** p < 0.0001, ** p < 0.01 and * p < 0.05: treated cells vs. respective untreated cells (Ctrl).

Article Snippet: Experiments were performed on the commercially available MPM cell line MSTO-211H (human biphasic mesothelioma cells), purchased from American Type Culture Collection (ATCC; Manassas, VA, USA), and primary MPM patient-derived cell lines MM404 (human epithelioid histotype), MM421 (human biphasic histotype) and MM432 (human sarcomatoid histotype) from Biological Bank of Mesothelioma (SS.

Techniques: Migration, Wound Healing Assay, Microscopy, Control, Transfection, Cell Cycle Assay, Flow Cytometry, Staining

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: Fig. 1 CRISPR screening of MPM and nonmalignant cell lines. A Schematic workflow of genome-wide loss-of-function CRISPR screens conducted in MeT-5A, H2052, H2452, and H28 cell lines. Cas9-expressing monoclonal cell lines were transduced with Brunello library at MOI ≌0.3 and selected with puromycin. Genomic DNA was extracted from T0 and T14 cell pellets. gRNA-targeted regions were amplified by PCR and sequenced on the HiSeq2500 system. Data were analyzed using MAGeCK software. High-confidence hits were identified based on negative CRISPR viability scores (depleted). B The dot plot showing the distributions of log2 normalized T14 gRNA read counts relative to T0 gRNA read counts. gRNAs targeting 360 core essential genes [88] (orange dots) and non-targeting gRNAs (blue dots), negative controls in the Brunello library, are shown. C Cellular and molecular pathways involving the genes with the highest depletion scores in the CRISPR screens were obtained by pathway enrichment analysis. D The log2 fold change of gRNAs for top-ranked and bottom-ranked 5 genes is depicted. The gRNAs with negative log2 fold change were represented as blue lines and the ones with positive log2 fold change were represented as red lines. E Correlation between our genome-wide CRISPR screen and CRISPR knockout screen data from the Project Achilles (DepMap Public 23Q2+Score, Chronos, access date: 09.06.2023). Genome-wide targets (gray) and fitness/essential genes (1580, orange) are highlighted. F ROC-AUC analysis of CRISPR knockout screening data across four cell lines. False positive rates are calculated based on nonessential genes and graphed in relation to the true positive rate, which is determined by essential genes.

Article Snippet: Cell Death and Disease (2025) 16:241 METHODS Cell lines and culture conditions Nonmalignant mesothelial cell line MeT-5A (CRL-9444) and MPM cell lines H2052 (NCI-H2052; CRL-5915), H2452 (NCI-H2452; CRL-5946), and H28 (NCI-H28; CRL-5820) were purchased from American Type Culture Collection (ATCC; Manassas, VA).

Techniques: CRISPR, Genome Wide, Expressing, Transduction, Software, 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: Fig. 7 BUB1 overexpression amplifies proliferative and malignant phenotypes. 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: Cell Death and Disease (2025) 16:241 METHODS Cell lines and culture conditions Nonmalignant mesothelial cell line MeT-5A (CRL-9444) and MPM cell lines H2052 (NCI-H2052; CRL-5915), H2452 (NCI-H2452; CRL-5946), and H28 (NCI-H28; CRL-5820) were purchased from American Type Culture Collection (ATCC; Manassas, VA).

Techniques: Over Expression, Plasmid Preparation, 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: Fig. 8 BUB1 is indispensable for maintaining SAC integrity and cytokinesis. A Heatmap depicting the expression levels of BUB1 local network genes (highlighted in Fig. S9A) 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: Cell Death and Disease (2025) 16:241 METHODS Cell lines and culture conditions Nonmalignant mesothelial cell line MeT-5A (CRL-9444) and MPM cell lines H2052 (NCI-H2052; CRL-5915), H2452 (NCI-H2452; CRL-5946), and H28 (NCI-H28; CRL-5820) were purchased from American Type Culture Collection (ATCC; Manassas, VA).

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

Journal: Translational Oncology

Article Title: The highly selective and oral phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib induces apoptosis in mesothelioma cells and increases immune effector cell composition

doi: 10.1016/j.tranon.2023.101857

Figure Lengend Snippet: Association between PI3K-δ expression and clinicopathological characteristics in 89 MPM patients.

Article Snippet: MPM patient-derived xenograft (PDX) cell lines PXF698 and PXF1118 (both from epithelioid tumors) and PXF1752 (from sarcomatoid MPM) were from Charles River Germany GmbH (Freiburg, Germany) and cultured in RPMI medium supplemented with 10% Fetal Bovine Serum (FBS) (Biochrom AG, Berlin, Germany).

Techniques: Expressing

Journal: Translational Oncology

Article Title: The highly selective and oral phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib induces apoptosis in mesothelioma cells and increases immune effector cell composition

doi: 10.1016/j.tranon.2023.101857

Figure Lengend Snippet: Roginolisib impairs mesothelioma cell viability and induces apoptotic cell death. (A) MPM cell lines PXF698, PXF1118 and PXF1752 were exposed to the indicated concentrations of roginolisib. The viability of the cells was measured after 72 h of treatment by a MTT cytotoxicity assay. Data points, mean ± SD of independent triplicate experiments. Immunohistochemistry analysis of PI3K-δ protein expression in PXF698, PXF1118 and PXF1752 cells. Scale, 50 µm. (B) PXF698 cells were exposed to roginolisib (100 µM), paclitaxel (400 nM) or DMSO in the presence of the RealTime-Glo Annexin V Apoptosis and Necrosis Assay. Luminescence (phosphatidylsprrerine:annexin V binding – apoptosis; solid line) and fluorescence (loss of membrane integrity – necrosis; dashed line) were recorded for 40 h at the indicated time points. A clear temporal lag between phosphatidylserine exposure and lack of membrane integrity is indicative of apoptotic cell death leading to secondary necrosis. Data points, mean of duplicate samples, representative of independent duplicate experiments. (C) Caspase-3/7 activity after treatment of cells with 100 µM roginolisib or 0.1% DMSO for 24 h. Data points, mean ± SD of independent triplicate experiments. 2way ANOVA test (Dunnett‘s multiple comparison); ****, p<0.0001; ***, p=0.0002. (D) Apoptotic membrane blebbing after treatment of PXF698 cells with 100 µM roginolisib for 24 h, detected by phase-contrast microscopy. (E) PARP cleavage and levels of Mcl-1 and BIM in PXF698 and PXF1752 cells after 100 µM roginolisib treatment at 0, 4, 24 h. (F) Immunoblotting shows the levels of p-AKT (S473) and p-ERK1/2 (T202/204) in PXF1118 and PXF1752 cells after 100 µM roginolisib treatment at 0, 4, 24 h. Bar graphs represent semi-quantitative analysis of phospho-protein expression relative to total protein, normalized to protein levels at 0 h. (G) Schematic illustration of PI3K/AKT/mTOR and ERK signaling.

Article Snippet: MPM patient-derived xenograft (PDX) cell lines PXF698 and PXF1118 (both from epithelioid tumors) and PXF1752 (from sarcomatoid MPM) were from Charles River Germany GmbH (Freiburg, Germany) and cultured in RPMI medium supplemented with 10% Fetal Bovine Serum (FBS) (Biochrom AG, Berlin, Germany).

Techniques: Cytotoxicity Assay, Immunohistochemistry, Expressing, Binding Assay, Fluorescence, Membrane, Activity Assay, Comparison, Microscopy, Western Blot

Journal: Translational Oncology

Article Title: The highly selective and oral phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib induces apoptosis in mesothelioma cells and increases immune effector cell composition

doi: 10.1016/j.tranon.2023.101857

Figure Lengend Snippet: Synergistic cytotoxicity of PI3K-δ inhibitors and sapanisertib (mTORC1/2 inhibitor) or ipatasertib (AKT inhibitor). (A) MPM cells were treated with PI3K-δ inhibitors roginolisib (Rog) or idelalisib (Idel) as single agents and in combination with sapanisertib (Sapa) (PXF698) or ipatasertib (Ipa) (PXF1118) for 72 h, after which cell viability was assessed using an MTT cytotoxicity assay. Data points, mean of quintuplets of two independent experiments. (B) Schematic illustration of PI3K/AKT/mTOR and ERK signaling. Growth factors and hormones activate PI3 kinase catalyzing the production of PIP3, which in turn coordinates cell proliferation, metabolism and survival via activating AKT and mTOR signaling pathways. mTOR kinase serves as core component of two distinct protein complexes, mTORC1 and mTORC2. Activated Ras triggers both PI3K/AKT and ERK signaling, regulating cell proliferation and survival. (C) Molecular effects of simultaneous PI3K-δ/mTOR and PI3K-δ/AKT inhibition. Immunoblotting shows the levels of p-AKT (S473), p-PRAS40 p-PRAS40 (T246), p-RPS6 (S235/236) and p-ERK1/2 (T202/204) in PXF1118 cells treated with 100 µM roginolisib (Rog), 100 µM idelalisib (Idel), 30 nM sapanisertib (Sapa), 10 µM ipatasertib (Ipa), or their combinations as indicated for 4 h.

Article Snippet: MPM patient-derived xenograft (PDX) cell lines PXF698 and PXF1118 (both from epithelioid tumors) and PXF1752 (from sarcomatoid MPM) were from Charles River Germany GmbH (Freiburg, Germany) and cultured in RPMI medium supplemented with 10% Fetal Bovine Serum (FBS) (Biochrom AG, Berlin, Germany).

Techniques: Cytotoxicity Assay, Protein-Protein interactions, Inhibition, Western Blot

Journal: Translational Oncology

Article Title: The highly selective and oral phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib induces apoptosis in mesothelioma cells and increases immune effector cell composition

doi: 10.1016/j.tranon.2023.101857

Figure Lengend Snippet: Roginolisib enhances the cytotoxicity of cisplatin and nivolumab in patient-derived tumor cells in the presence of stromal cells. (A) BAP1-wild-type epithelioid (BAP + epi) and BAP1-null sarcomatoid (BAP − sar) MPM cells were co-cultured with peripheral mononuclear cells (PBMCs) and exposed to the indicated concentrations of roginolisib in the absence (-MRC5) or presence of fibroblasts (+MRC5). The viability of the cells was measured after 72 hours by WST-1 staining. Data points, mean of independent quadruplicate experiments. (B) MPM cells were treated with 5 µM cisplatin (Pt) and 1 µg/ml nivolumab (Nivo) for 72 h in the absence and presence of MRC5 cells. (C, D) The combination of roginolisib with 5 µM cisplatin plus 1 µg/ml nivolumab, either as combined/concomitant treatment (C) or in sequential treatment (D), was investigated in the absence (-MRC5) and presence of fibroblasts (+MRC5). 0: untreated cells. Nonparametric Kruskal–Wallis test followed by Dunn's multiple comparison test; ***p<0.001: vs. untreated cells; °p<0.05, °°°p<0.001: MRC5+ vs MRC5-.

Article Snippet: MPM patient-derived xenograft (PDX) cell lines PXF698 and PXF1118 (both from epithelioid tumors) and PXF1752 (from sarcomatoid MPM) were from Charles River Germany GmbH (Freiburg, Germany) and cultured in RPMI medium supplemented with 10% Fetal Bovine Serum (FBS) (Biochrom AG, Berlin, Germany).

Techniques: Derivative Assay, Cell Culture, Staining, Comparison

Journal: Translational Oncology

Article Title: The highly selective and oral phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib induces apoptosis in mesothelioma cells and increases immune effector cell composition

doi: 10.1016/j.tranon.2023.101857

Figure Lengend Snippet: Roginolisib exerts immunomodulatory activity. BAP1-wild-type epithelioid (A) and BAP1-null sarcomatoid (B) MPM cells were co-cultured with peripheral mononuclear cells (PBMCs) in the absence or presence of MRC5 fibroblasts and treated with roginolisib alone, co-incubated with cisplatin/nivolumab or in sequential treatment (cisplatin/nivolumab followed by roginolisib). The immunophenotype of immune cells was analyzed by flow cytometry (n = 2, two independent experiments). *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: MPM patient-derived xenograft (PDX) cell lines PXF698 and PXF1118 (both from epithelioid tumors) and PXF1752 (from sarcomatoid MPM) were from Charles River Germany GmbH (Freiburg, Germany) and cultured in RPMI medium supplemented with 10% Fetal Bovine Serum (FBS) (Biochrom AG, Berlin, Germany).

Techniques: Activity Assay, Cell Culture, Incubation, Flow Cytometry