set 2 cell lines  (DSMZ)

Journal: Frontiers in immunology

Article Title: Platelet-derived microparticles provoke chronic lymphocytic leukemia malignancy through metabolic reprogramming.

doi: 10.3389/fimmu.2023.1207631

Figure Lengend Snippet: FIGURE 2 Impact of mitochondria acquisition on CLL viability. CLL cell lines were evaluated for cell viability at varying co-culture ratios (1:10 and 1:100) and incubation time (24 and 48 hours) with either (A) PMPs or (B) purified mitochondria isolated from the Set2 megakaryocytic cell line. Viability was established using a cell-based colorimetric assay (Cell Titer-Blue, Promega) combined a multi-well plate spectral analysis from a Synergy H1 Hybrid Multi-Mode Microplate Reader (Ex/Em: 560/590 nm). Each value is the mean ± SEM of six separate experiments. The asterisk (*) indicates significantly different (*p < 0.05, ***p < 0.001). (C) Light microscopy (40X) of CLLs co-incubations with PMPs or Set2-derived mitochondria was also performed for morphological examination.

Article Snippet: Human leukemic cell lines CII (#ACC 773), MEC-1 (ACC 497), and the megakaryocytic Set2 cell line (#ACC 608) were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture (DSMZ Braunschweig, Germany).

Techniques: Co-Culture Assay, Incubation, Isolation, Colorimetric Assay, Light Microscopy, Derivative Assay

Journal: Frontiers in immunology

Article Title: Platelet-derived microparticles provoke chronic lymphocytic leukemia malignancy through metabolic reprogramming.

doi: 10.3389/fimmu.2023.1207631

Figure Lengend Snippet: FIGURE 3 Impact of PMPs on CLL metabolic functions. CLL cell lines (CII and MEC-1) were evaluated for metabolic functions at varying co-culture ratios (1:10 and 1:100) and incubation periods (24 and 48 hours) with either PMPs or purified mitochondria isolated from the Set2 megakaryocytic cell line. (A) Oxygen consumption rate (OCR) was determined using an Oroboros-O2k for: ROUTINE respiration as the basal state; the non-phosphorylating resting state (LEAK respiration); and the electron transport system (ETS) capacity. (B) Total ATP and glycolytic ATP levels were evaluated using a firefly luciferase-based assay. Relative ATP concentrations related to specific pathways (oxidative phosphorylation/OXPHOS versus glycolysis) were determined by cell treatments to inhibit mitochondrial complexes I and III using Rotenone (ROT) and Antimycin A (AmA), respectively. (C, D) Cells were treated with AmA, ROT, and phorbol 12-myristate 13-acetate (PMA) as ROS inducers. Thereafter, samples were mixed with (C) CellROX green reagent and SYTOX Red Dead Cell Stain prior analysis by flow cytometry at Ex/Em: 508/525 nm (CellROX green) and 640/658 nm (SYTOX Red Dead Cell Stain) to determine intracellular total ROS content; or with (D) MitoSOX™reagent prior analysis by flow cytometry at Ex/Em: 510/580 nm to determine mitochondrial superoxide content. N-acetylcysteine (NAC) and Tert-butyl hydroperoxide (TBHP) were used as negative and positive controls, respectively. Results are expressed as the mean ± SEM of six biological experiments. One-way ANOVA followed by Tukey’s multiple comparisons test show significant differences in the values presenting different superscript letters (p < 0.05).

Article Snippet: Human leukemic cell lines CII (#ACC 773), MEC-1 (ACC 497), and the megakaryocytic Set2 cell line (#ACC 608) were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture (DSMZ Braunschweig, Germany).

Techniques: Co-Culture Assay, Incubation, Isolation, Luciferase, Phospho-proteomics, Staining, Cytometry

Journal: Blood cancer journal

Article Title: Targeting PP2A-dependent autophagy enhances sensitivity to ruxolitinib in JAK2 V617F myeloproliferative neoplasms.

doi: 10.1038/s41408-023-00875-x

Figure Lengend Snippet: Fig. 1 Ruxolitinib increases autophagy in JAK2V617F cells. A, B HEL and SET-2 cells were treated with ruxolitinib (1 µM) for 2 hours in the presence or absence of bafilomycin (25 nM) to monitor the autophagy flux. A Cells were processed for western blot analysis of P-STAT5, STAT5 and LC3B. Actin was used as a loading control. Graphs represent the LC3B-II/actin ratio obtained by densitometric analysis (n = 4 ± sem). B Cells were stained for LC3B and analyzed by confocal microscopy. Graphs represent the number of LC3B dots per cell (n = 4 ± sem). Scale bar: 10 µm. C Mononuclear cells from peripheral blood samples from JAK2V617F-positive MPN patients were separated by Ficoll-Hypaque density gradient centrifugation. CD34+ progenitors were then sorted using an immunomagnetic positive selection kit and processed for further experiments. D Patients’ cells obtained in C were treated or not with chloroquine (20 µM) over 16 hours to monitor autophagy flux in combination with ruxolitinib (1 µM) for 2 h and stained for LC3B. Representative confocal pictures are shown and histograms represent the number of LC3B dots per cell (n = 8 ± sem). Scale bar: 10 µm. E, F JAK2WT HL-60 cells were treated with ruxolitinib (1 µM) for 2 hours in the presence or absence of bafilomycin (25 nM) to monitor autophagy flux. E Cells were processed for western blot analysis of P-STAT5, STAT5 and LC3B. Actin was used as a loading control. Graphs represent the LC3B-II/actin ratio obtained by densitometric analysis (n = 3 ± sem). F Cells were stained for LC3B and analyzed by confocal microscopy. Graphs represent the number of LC3B dots per cell (n = 3). Scale bar: 10 µm.

Article Snippet: MATERIALS AND METHODS Cell lines culture and treatments The human cell lines HEL (expressing JAK2V617F), SET-2 (expressing both JAK2V617F and JAK2WT) and HL-60 (expressing JAK2WT) were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).

Techniques: Western Blot, Control, Staining, Confocal Microscopy, Gradient Centrifugation, Selection