Journal: Thoracic Cancer

Article Title: Effects of Autophagy Inhibition by SAR405 , a Selective VPS34 Inhibitor, on Pleural Mesothelioma Cells

doi: 10.1111/1759-7714.70255

Figure Lengend Snippet: Effects of SAR405 on colony formation by mesothelioma cells. (A) Cells were spread on dishes and cultured for 10 days in 211H and for 14 days in H28 and H2452 with 2.5–5.0 μM SAR405 or dimethyl sulfoxide (DMSO). (B) Mean (±SD) number of colonies formed by each mesothelioma cell line. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with DMSO.

Article Snippet: Human pleural mesothelioma cell lines 211H, H2452, and H28 (all from American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI 1640 medium (SH30027.01; GE HealthCare, Tokyo, Japan) supplemented with 10% fetal bovine serum (Sigma‐Aldrich, St. Louis, MO, USA) and 1% penicillin–streptomycin (168–23 191; FUJIFILM Wako Pure Chemical, Osaka, Japan) at 37°C under 5% CO2.

Techniques: Cell Culture

Journal: Thoracic Cancer

Article Title: Effects of Autophagy Inhibition by SAR405 , a Selective VPS34 Inhibitor, on Pleural Mesothelioma Cells

doi: 10.1111/1759-7714.70255

Figure Lengend Snippet: Effects of SAR405 on cell migration/invasion. (A) Cell invasion of H28, H2452, and 211H mesothelioma cells was measured using a scratch wound healing assay. A single scratch was made in the center of the confluent cell monolayer, and cells were then treated with 10.0 μM SAR405 or dimethyl sulfoxide (DMSO) as a control. Cell invasion was monitored for 48 h and visualized by light microscopy. (B) Wound area remaining at 48 h in cells treated with DMSO or 10.0 μM SAR405. Data are the mean ± SD of three independent experiments. ** p < 0.01, *** p < 0.001 compared with DMSO.

Article Snippet: Human pleural mesothelioma cell lines 211H, H2452, and H28 (all from American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI 1640 medium (SH30027.01; GE HealthCare, Tokyo, Japan) supplemented with 10% fetal bovine serum (Sigma‐Aldrich, St. Louis, MO, USA) and 1% penicillin–streptomycin (168–23 191; FUJIFILM Wako Pure Chemical, Osaka, Japan) at 37°C under 5% CO2.

Techniques: Migration, Wound Healing Assay, Control, Light Microscopy

Journal: Thoracic Cancer

Article Title: Effects of Autophagy Inhibition by SAR405 , a Selective VPS34 Inhibitor, on Pleural Mesothelioma Cells

doi: 10.1111/1759-7714.70255

Figure Lengend Snippet: Effects of SAR405 on the cell cycle in H28, H2452, and 211H mesothelioma cells. SAR405 induced G 2 /M cycle arrest in pleural mesothelioma cells: The proportion of cells in the G 2 /M phase was higher after culture with 2.5–10.0 μM SAR405 for 48 h than after culture with dimethyl sulfoxide (DMSO) as the control.

Article Snippet: Human pleural mesothelioma cell lines 211H, H2452, and H28 (all from American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI 1640 medium (SH30027.01; GE HealthCare, Tokyo, Japan) supplemented with 10% fetal bovine serum (Sigma‐Aldrich, St. Louis, MO, USA) and 1% penicillin–streptomycin (168–23 191; FUJIFILM Wako Pure Chemical, Osaka, Japan) at 37°C under 5% CO2.

Techniques: Control

Journal: Thoracic Cancer

Article Title: Effects of Autophagy Inhibition by SAR405 , a Selective VPS34 Inhibitor, on Pleural Mesothelioma Cells

doi: 10.1111/1759-7714.70255

Figure Lengend Snippet: Effects of cisplatin on autophagy in pleural mesothelioma cells. (A) Fluorescence microscopy findings for transfected mesothelioma cells cultured for 24 h with 0.5–5.0 μM cisplatin or control (normal saline). Scale bars = 200 μm. (B) Signal intensities of green fluorescent protein (GFP) and red fluorescent protein (RFP) were measured by flow cytometry after 24 h culture of H28, H2452, and 211H cells with 0.5–5.0 μM cisplatin or control (normal saline), and the GFP/RFP ratio was determined. Data are the mean ± SD of three independent experiments. ** p < 0.01, *** p < 0.001 compared with control.

Article Snippet: Human pleural mesothelioma cell lines 211H, H2452, and H28 (all from American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI 1640 medium (SH30027.01; GE HealthCare, Tokyo, Japan) supplemented with 10% fetal bovine serum (Sigma‐Aldrich, St. Louis, MO, USA) and 1% penicillin–streptomycin (168–23 191; FUJIFILM Wako Pure Chemical, Osaka, Japan) at 37°C under 5% CO2.

Techniques: Fluorescence, Microscopy, Transfection, Cell Culture, Control, Saline, Flow Cytometry

Journal: Thoracic Cancer

Article Title: Effects of Autophagy Inhibition by SAR405 , a Selective VPS34 Inhibitor, on Pleural Mesothelioma Cells

doi: 10.1111/1759-7714.70255

Figure Lengend Snippet: No synergistic effect was seen for the combination of SAR405 and cisplatin. (A) Mesothelioma cells were cultured for 72 h with 1.0–5.0 μM cisplatin, 2.5–10.0 μM SAR405, or dimethyl sulfoxide (DMSO) as a control. Cell viability was assessed by the reduction of water‐soluble tetrazolium 8 (WST‐8). The results show the proportion of viable cells after 72 h treatment with either cisplatin combined with SAR405 or DMSO. The proportion of viable cells was lower after combined treatment with cisplatin and SAR405 than after treatment with DMSO. Data are the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control. (B) Drug interactions of cisplatin with either SAR405, PIK‐III, or Vps34‐IN‐1 in mesothelioma cell lines. H28, H2452, and 211H cells were cultured for 72 h with 0.1–100 μM cisplatin and 0.1–100 μM SAR405, PIK‐III, or Vps34‐IN‐1. Drug interactions were evaluated using SynergyFinder (version 3.0) using the zero interaction potency (ZIP) model. A ZIP synergy score > 10 is considered synergistic; a score between −10 and 10 is considered additive; and a score less than −10 is considered antagonistic.

Article Snippet: Human pleural mesothelioma cell lines 211H, H2452, and H28 (all from American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI 1640 medium (SH30027.01; GE HealthCare, Tokyo, Japan) supplemented with 10% fetal bovine serum (Sigma‐Aldrich, St. Louis, MO, USA) and 1% penicillin–streptomycin (168–23 191; FUJIFILM Wako Pure Chemical, Osaka, Japan) at 37°C under 5% CO2.

Techniques: Cell Culture, Control

Journal: Antibodies

Article Title: Ca 13 Mab-17, a Novel Anti-Cadherin-13 Monoclonal Antibody for Versatile Applications

doi: 10.3390/antib15030039

Figure Lengend Snippet: Flow cytometric analysis of Ca 13 Mab-17 for detection of endogenous CDH13. LN229 ( A ), U87MG ( B ), and NCI-H2052 ( C ) were treated with Ca 13 Mab-17 at the indicated concentrations (red) or with blocking buffer (black, negative control). The Ca 13 Mab-17-treated cells were incubated with Alexa Fluor 488-conjugated anti-mouse IgG. Fluorescence data were collected using the SA3800 Cell Analyzer.

Article Snippet: Chinese hamster ovary (CHO)-K1, mouse myeloma P3X63Ag8U.1 (P3U1), human glioblastoma (GBM) LN229, U87MG, and human mesothelioma NCI-H2052 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Blocking Assay, Negative Control, Incubation, Fluorescence

Journal: Life

Article Title: Quercetin–Arctigenin Co-Treatment Induces Mitochondrial Dysfunction and Apoptotic Cell Death Through Metabolic Stress in Malignant Mesothelioma Cells

doi: 10.3390/life16050774

Figure Lengend Snippet: Effects of quercetin and arctigenin on relative metabolic activity in mesothelioma and mesothelial cells. ( A – C ) MSTO-211H and MeT-5A cells were treated with increasing concentrations of QUE or ATG for ( A ) 24 h, ( B ) 48 h, and ( C ) 72 h. Relative metabolic activity was measured using the MTT assay. Data are presented as the mean ± SD from at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test (* p < 0.05 vs. control).

Article Snippet: Human malignant mesothelioma MSTO-211H cells and human mesothelial MeT-5A cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Activity Assay, MTT Assay, Control

Journal: Life

Article Title: Quercetin–Arctigenin Co-Treatment Induces Mitochondrial Dysfunction and Apoptotic Cell Death Through Metabolic Stress in Malignant Mesothelioma Cells

doi: 10.3390/life16050774

Figure Lengend Snippet: Combined treatment with quercetin and arctigenin reduces metabolic activity and intracellular ATP production in mesothelioma cells. ( A – C ) MSTO-211H and MeT-5A cells were treated with QUE (40 μM), ATG (50 μM), or their combination for ( A ) 24 h, ( B ) 48 h, and ( C ) 72 h. Relative metabolic activity was measured using the MTT assay. ( D – F ) Intracellular ATP levels were measured after ( D ) 24 h, ( E ) 48 h, and ( F ) 72 h treatment. Data are presented as the mean ± SD from at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test (* p < 0.05, *** p < 0.001, **** p < 0.0001 vs. control).

Article Snippet: Human malignant mesothelioma MSTO-211H cells and human mesothelial MeT-5A cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Activity Assay, MTT Assay, Control

Journal: Life

Article Title: Quercetin–Arctigenin Co-Treatment Induces Mitochondrial Dysfunction and Apoptotic Cell Death Through Metabolic Stress in Malignant Mesothelioma Cells

doi: 10.3390/life16050774

Figure Lengend Snippet: Combination treatment suggests a potential synergistic interaction, reduces cell growth capacity, and increases caspase activity in mesothelioma cells. ( A ) Combination index (CI) values were calculated for QUE (40 μM) and ATG (50 μM) co-treatment in MeT-5A and MSTO-211H cells at 24, 48, and 72 h. CI values < 1 may be indicative of a potential synergistic interaction under the experimental conditions tested. ( B ) Cell growth capacity was assessed by crystal violet staining following 48 h treatment with QUE, ATG, or their combination. ( C ) Caspase 3/7 activity was measured after 48 h of treatment to evaluate apoptosis-associated protease activation. Data are presented as the mean ± SD from at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control).

Article Snippet: Human malignant mesothelioma MSTO-211H cells and human mesothelial MeT-5A cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Activity Assay, Staining, Activation Assay, Control

Journal: Life

Article Title: Quercetin–Arctigenin Co-Treatment Induces Mitochondrial Dysfunction and Apoptotic Cell Death Through Metabolic Stress in Malignant Mesothelioma Cells

doi: 10.3390/life16050774

Figure Lengend Snippet: Combined treatment with quercetin and arctigenin suggests increased apoptotic cell populations in mesothelioma cells. Representative Annexin V dot plots showing apoptotic cell populations in MeT-5A and MSTO-211H cells following treatment with QUE (40 μM), ATG (50 μM), or their combination for 48 h. Dot plots represent cells stained with Annexin V and PI, where color intensity indicates cell density and quadrants correspond to live, early apoptotic, and late apoptotic cell populations. Percentages of live, early apoptotic, and late apoptotic cells are indicated. Lower panels show quantitative analysis of live cells (%) and total apoptotic cells (%). Data are presented as the mean ± SD from at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test (** p < 0.01, **** p < 0.0001 vs. control).

Article Snippet: Human malignant mesothelioma MSTO-211H cells and human mesothelial MeT-5A cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Staining, Control

Journal: Life

Article Title: Quercetin–Arctigenin Co-Treatment Induces Mitochondrial Dysfunction and Apoptotic Cell Death Through Metabolic Stress in Malignant Mesothelioma Cells

doi: 10.3390/life16050774

Figure Lengend Snippet: Co-treatment with quercetin and arctigenin is associated with modulation of mitochondrial apoptosis-associated signaling and energy stress-related protein expression in mesothelioma cells. ( A ) Representative Western blot analysis of apoptosis-associated proteins in MeT-5A and MSTO-211H cells treated with quercetin (QUE, 40 μM), arctigenin (ATG, 50 μM), or their combination for 48 h. Expression levels of anti-apoptotic proteins Mcl-1, Bcl-xL, and Bcl-2, as well as apoptosis execution markers cleaved caspase-3 and cleaved PARP, were examined. β-actin was used as the loading control. ( B ) Representative Western blot analysis of energy stress- and mitochondrial protein expression-related markers. Expression levels of phosphorylated AMPK (p-AMPK), total AMPK, and selected oxidative phosphorylation (OXPHOS) complex subunits (ATP5A, UQCRC2, SDHB, COX II, and NDUFB8) were evaluated in MeT-5A and MSTO-211H cells following treatment with QUE, ATG, or their combination for 48 h. Relative protein expression levels were normalized to β-actin, and the p-AMPK/AMPK ratio was calculated to evaluate AMPK activation. Densitometric values are presented as mean ± SD from three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control). Faint background signals may be present due to exposure sonditions and do not affect the interpretation of the results.

Article Snippet: Human malignant mesothelioma MSTO-211H cells and human mesothelial MeT-5A cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Western Blot, Control, Phospho-proteomics, Activation Assay

Journal: Life

Article Title: Quercetin–Arctigenin Co-Treatment Induces Mitochondrial Dysfunction and Apoptotic Cell Death Through Metabolic Stress in Malignant Mesothelioma Cells

doi: 10.3390/life16050774

Figure Lengend Snippet: Proposed mechanism underlying the cooperative effects of quercetin and arctigenin in mesothelioma cells. Schematic illustration of the proposed mechanism by which co-treatment with quercetin (QUE) and arctigenin (ATG) reduces mesothelioma cell viability through induction of metabolic stress and apoptosis-associated signaling pathways. Combined treatment decreases intracellular ATP production and activates AMPK signaling, accompanied by altered mitochondrial OXPHOS protein expression patterns. These metabolic alterations are associated with reduced expression of anti-apoptotic proteins Mcl-1, Bcl-2, and Bcl-xL, leading to activation of caspase-dependent apoptotic signaling, as evidenced by increased cleaved caspase-3 and cleaved PARP, ultimately contributing to apoptotic cell death in MSTO-211H mesothelioma cells. Arrows indicate the direction of signaling interactions, while upward and downward arrows represent increases and decreases in protein expression or activity, respectively.

Article Snippet: Human malignant mesothelioma MSTO-211H cells and human mesothelial MeT-5A cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Activity Assay