Journal: Oncotarget

Article Title: Resistance to glucose starvation as metabolic trait of platinum-resistant human epithelial ovarian cancer cells

doi: 10.18632/oncotarget.14118

Figure Lengend Snippet: ( A ) Flow cytometry analysis of cell viability in ascitic effusion cells isolated from 47 EOC patients categorized as PLT-sensitive/ resistant on the basis of their clinical response to PLT treatment. Cell viability of CD45 neg /CD44 pos tumor cells was recorded after 14-day in vitro culture in the presence (glucose) and in the absence (no glucose) of glucose by Live/Dead staining. Each circle represents an individual patient, and the dotted line indicates the median value (13.0%), chosen as cut-off value. ( B ) The bar graph summarizes mean values (± SD) of tumor cells from EOC patients categorized as glucose addicted (GA) or glucose non-addicted (GNA) according to their cell viability after 14 d under glucose starvation. * P < 0.05. ( C ) Cell viability analysis of AnnexinV/PI staining in CD45 neg /CD44 pos tumor cells from GA ( n = 6) and GNA ( n = 6) patients treated in vitro for 72 h with different doses of carboplatin (2.5, 5 and 10 μg/ml). Data were expressed as mean percent viability (± SD) compared to control. The LD50 value for GA and GNA samples is indicated on the abscissa axis. ( D ) Flow cytometry analysis of cell morphology and viability by AnnexinV/PI staining in CD45 neg /CD44 pos tumor cells from two representative GA and GNA patients, treated in vitro for 72 h with different carboplatin concentrations. ( E ) WB analysis of total and cleaved PARP in FACS-sorted CD45 neg /CD44 pos tumor cells of GA and GNA patients, treated in vitro for 72 h with different doses of carboplatin. One representative blot is shown on the left panel; the right histogram depicts mean values (± SD) of 6 GA vs 6 GNA patients. Signal intensities of the PARP bands were normalized against the α-tubulin signal. Expression ratios were calculated by dividing normalized signal intensity values obtained for untreated GA or GNA cells. * P < 0.05.

Article Snippet: For flow cytometry analysis, cells were stained with Live/Dead and anti-CD44 (Abcam), CD45 (Miltenyi), CD117 (1:100, Miltenyi Biotec) and GLUT1 (1:1000, Abcam) antibodies.

Techniques: Flow Cytometry, Isolation, In Vitro, Staining, Control, Expressing

Journal: Oncotarget

Article Title: Resistance to glucose starvation as metabolic trait of platinum-resistant human epithelial ovarian cancer cells

doi: 10.18632/oncotarget.14118

Figure Lengend Snippet: ( A ) Flow cytometry evaluation of membrane GLUT1. Data expressed as mean percent values in tumor cells from GA and GNA patients (± SD). * P < 0.05. ( B ) qRT-PCR analysis of key enzymes of the glucose metabolic chain in FACS sorted CD45 neg /CD44 pos tumor cells from GA ( n = 6) and GNA ( n = 6) patients. * P < 0.01. ( C ) ECAR analysis in CD45 neg /CD44 pos tumor cells from GA and GNA patients. The left histogram shows the mean values of the basal ECAR in 6 GA vs 6 GNA patients (± SD). * P < 0.05. One representative experiment is shown on the right; the first three points of the graph indicate the basal ECAR ratio in glucose starvation. At different time points (arrows) the indicated inhibitors of OXPHOS (oligomycin) or glycolysis (2DG) were added. ( D ) WB analysis of MCT4 lactate transporter in pooled CD45 neg /CD44 pos tumor cells from GA or GNA patients (pools #1) and xenotransplants (pools #2). Each pool consisted of 3 GA or 3 GNA samples. After normalization against actin, expression ratios were calculated by dividing GNA signal intensity values by those of GA cells. On the left a representative blot, on the right graph depicts mean expression ratios in 8 different pools (± SD). * P < 0.05. ( E ) WB analysis of Beclin1and LC3-II proteins in CD45 neg /CD44 pos tumor cell pools of GA or GNA patients. One representative blot is shown on the left, while the right histogram shows the mean values of 4 different experiments (three samples/pool; ± SD). * P < 0.05. ( F ) Left panel: flow cytometry analysis of cell morphology and viability by AnnexinV/PI staining in two representative GA and GNA samples, treated in vitro for 72 h with 20 μM CQ or 100 nM BafA1. Right panel: histogram displaying the mean fold change (± SD) in cell viability, calculated by dividing the percentage of live cells after treatment by untreated values of 4 GA and 5 GNA samples. * P < 0.05.

Article Snippet: For flow cytometry analysis, cells were stained with Live/Dead and anti-CD44 (Abcam), CD45 (Miltenyi), CD117 (1:100, Miltenyi Biotec) and GLUT1 (1:1000, Abcam) antibodies.

Techniques: Flow Cytometry, Membrane, Quantitative RT-PCR, Expressing, Staining, In Vitro

Journal: Oncotarget

Article Title: Resistance to glucose starvation as metabolic trait of platinum-resistant human epithelial ovarian cancer cells

doi: 10.18632/oncotarget.14118

Figure Lengend Snippet: ( A ) The proliferation rate of GA vs. GNA ascitic effusion cells was evaluated by CFSE staining in CD45 neg /CD44 pos tumor cells. Three gates were set according to dye intensity (time 0; left panel). For each gate, the mean percentage of CFSE + cells was quantified 72 h after staining. One representative experiment is shown on the left; mean values (± SD) in 5 GA and 4 GNA patients are plotted on the right. * P < 0.05. ( B ) qRT-PCR analysis of cyclin expression in FACS sorted CD45 neg /CD44 pos tumor cells from EOC ascitic effusions. Data were expressed as mean relative expression values in tumor cells from GA ( n = 6) compared to GNA ( n = 6) patients (± SD). * P < 0.001. ( C ) Left panel: Representative WB analysis of Cyclin D expression in pools of CD45 neg /CD44 pos tumor cells from GA and GNA patients (pools #1) and patient-derived xenotransplants (pools #2). Each pool consisted of 3 GA or GNA samples. Right panel: The signal intensity of the Cyclin D bands was normalized against the actin signal; expression ratios were calculated by dividing normalized signal intensity values obtained for GNA cells by those obtained for GA cells. The graph shows mean expression ratios (± SD) in 8 different pools. * P < 0.05. ( D ) qRT-PCR analysis of multidrug resistance (MDR) pump expression in FACS sorted CD45 neg /CD44 pos ascitic effusion cells from GA ( n = 6) and GNA ( n = 6) patients. Data were expressed as mean relative expression values (± SD). * P < 0.05. ( E ) Ex vivo flow cytometry analysis of ALDH activity in CD45 neg /CD44 pos EOC ascitic effusion cells from GA ( n = 20) and GNA ( n = 15) patients. Gates were set on the isotype control, and values indicate the percentage of ALDH +pos cells. One representative experiment is shown on the left; the right histogram shows mean percent values (± SD). * P < 0.05.

Article Snippet: For flow cytometry analysis, cells were stained with Live/Dead and anti-CD44 (Abcam), CD45 (Miltenyi), CD117 (1:100, Miltenyi Biotec) and GLUT1 (1:1000, Abcam) antibodies.

Techniques: Staining, Quantitative RT-PCR, Expressing, Derivative Assay, Ex Vivo, Flow Cytometry, Activity Assay, Control

Journal: Oncotarget

Article Title: Resistance to glucose starvation as metabolic trait of platinum-resistant human epithelial ovarian cancer cells

doi: 10.18632/oncotarget.14118

Figure Lengend Snippet: ( A ) Cell viability analysis performed with AnnexinV/PI staining in PLT-sensitive EOC xenografts (#S) and their resistant counterpart (#R) after 72 h of carboplatin treatment. Data are expressed as ratio between treated and untreated counterpart. ( B ) ALDH activity analysis in PLT-sensitive EOC xenografts (#S) and their resistant counterpart (#R) obtained as described in Materials and Methods. On the left one representative sample is shown; the right histogram shows mean values (± SD) of three biological replicates for two different samples. ( C ) Cell viability analysis of PLT-sensitive EOC xenografts (#S) and their resistant counterpart (#R) recorded after 7-day in vitro culture in the presence (glucose) and in the absence (no glucose) of glucose by Live/Dead staining. ( D ) qRT-PCR analysis of key enzymes involved in the glucose metabolic chain in PLT-sensitive EOC xenografts (#S) and their resistant counterpart (#R). Data are expressed as mean relative expression values (± SD). * P < 0.05. ( E ) Flow cytometry analysis of cell viability after 14 d in the presence or absence of glucose. The graph summarizes mean values (± SD) of tumor cells isolated from 23 EOC samples of untreated patients categorized as GA or GNA according to their cell viability under glucose starvation. * P < 0.05. ( F ) Kaplan-Meier curve showing the association between the GA/GNA phenotype and progression-free survival in a cohort of untreated EOC patients.

Article Snippet: For flow cytometry analysis, cells were stained with Live/Dead and anti-CD44 (Abcam), CD45 (Miltenyi), CD117 (1:100, Miltenyi Biotec) and GLUT1 (1:1000, Abcam) antibodies.

Techniques: Staining, Activity Assay, In Vitro, Quantitative RT-PCR, Expressing, Flow Cytometry, Isolation

Journal: Journal of Extracellular Biology

Article Title: Flow Cytometric Detection of Biomarker Changes in CFDA‐SE‐Labelled Plasma Extracellular Vesicles Using a Rodent Pregnancy Model of Prenatal Diagnostics

doi: 10.1002/jex2.70145

Figure Lengend Snippet: Changes in tetraspanin display in plasma EVs during pregnancy . (a–d) Flow cytometry data. Graphs to show proportion of CFDA‐SE positive particles expressing each biomarker. Each spot represents the mean of three technical replicates for one biological replicate, and the bars represent the group mean and standard deviation. p values indicate result of a t test. All samples are normally distributed (Shapiro–Wilk test) and of equal variance (Levene test). (e, f) ROC curves derived from the data shown in (a–d). (a, e) CD63 (B). (b, f) CD63 (M). (c, g) CD81. (d, h) CD9. CFDA‐SE, carboxyfluorescein diacetate succinimidyl ester; EV, extracellular vesicle; ROC, receiver operator characteristic.

Article Snippet: CD63‐M , Monoclonal IgG1 (recombinant) , Human , APC , Miltenyi 130‐134‐122 , Miltenyi 130‐113‐446.

Techniques: Clinical Proteomics, Flow Cytometry, Expressing, Biomarker Discovery, Standard Deviation, Derivative Assay