Journal: Oncotarget

Article Title: Dasatinib modulates sensitivity to pemetrexed in malignant pleural mesothelioma cell lines

doi: 10.18632/oncotarget.10428

Figure Lengend Snippet: In all MPM cell lines analyzed, the subcellular distribution of both TS and pSRC was detected by confocal microscopy after immunostaining with anti-TS (FITC-green, left panel) and anti-pSRC (FITC-green, right panel). Propidium iodide was used to counter stain nuclei, and the images were overlaid to determine both TS and pSRC localization within the cell. Confocal series images were taken on an inverted Zeiss LSM510 microscope equipped with a Plan-Apochromat 63X/1.4 oil immersion Ph 3 objective (Oberkochen, Germany). Scale bars: 10 μm. ( A ). Basal TS and SRC genes expression was determined by means of quantitative Real Time PCR ( B ) and TS, SRC and pSRC protein expression was also detected using immunoblotting ( C ). BACT served as housekeeping control.

Article Snippet: Three human MPM cell lines (MSTO-211H, NCI-MPP89, and REN) were used: MSTO-211H (designated MSTO) and MPP89 were purchased from Interlab Cell Line Collection (Genova, Italy), while REN [ ] cells were kindly provided by Dr. Moro (University of Novara, Italy) and Dr. Albelda (University of Pennsylvania, Philadelphia, USA).

Techniques: Confocal Microscopy, Immunostaining, Staining, Microscopy, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Oncotarget

Article Title: Dasatinib modulates sensitivity to pemetrexed in malignant pleural mesothelioma cell lines

doi: 10.18632/oncotarget.10428

Figure Lengend Snippet: PEM and dasatinib efficacy was established in all three MPM cell lines. REN and MPP89 (left and middle panels) were incubated with single and combination drugs for 72 hours, while 48 hours were sufficient for MSTO (right panel) ( A ). The apoptotic rate displayed differential responses, among MPM cells, to single and double treatments after 72 (left and middle graph) and 48 (right graph) hours, respectively ( B ). The same incubation times were applied to detect TS, SRC and pSRC protein expression ( C ). Wound healing assay was employed to determine the effect mediated by drugs on cell migration. Compared with untreated, dasatinib treatment abolished cell migration capability to heal the scrape after 24 hours in both REN and MPP89 cell lines (left and middle panels), and after 8 hours in MSTO (right panel). Plots display the progressive time-depending wound healing, in pixel units (px), under different drugs conditions ( D ).

Article Snippet: Three human MPM cell lines (MSTO-211H, NCI-MPP89, and REN) were used: MSTO-211H (designated MSTO) and MPP89 were purchased from Interlab Cell Line Collection (Genova, Italy), while REN [ ] cells were kindly provided by Dr. Moro (University of Novara, Italy) and Dr. Albelda (University of Pennsylvania, Philadelphia, USA).

Techniques: Incubation, Expressing, Wound Healing Assay, Migration

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