human cell lines sem  (DSMZ)

Journal: Cell Death Discovery

Article Title: Effective tumor cell abrogation via Venetoclax-mediated BCL-2 inhibition in KMT2A -rearranged acute B-lymphoblastic leukemia

doi: 10.1038/s41420-022-01093-3

Figure Lengend Snippet: A Dose-response curves (nonlinear regression, curve fit) of cell lines SEM, RS4;11, REH, and NALM-6 were incubated with increasing concentrations of VEN for 72 h. Proliferation and metabolic activity were assessed by trypan blue staining and WST-1 assay, respectively. Mean ± SD of 1–3 biological replicates. B Erythrocyte and PBMC cytotoxicity were evaluated by hemolysis and calcein-AM assay, respectively. The blood of five healthy donors was used. Mean ± SD of three technical replicates. Hemolytic activity was assessed by hemoglobin release after 120 min incubation with 10 nM VEN or 1% SDS (positive control). For viability testing, PBMCs were incubated with 10 nM VEN or DMSO (control) for 24 h. C Cells were treated with 2.5 nM (RS4;11) or 10 nM (SEM, PBMCs) VEN for 48 h and subsequently spun onto microscopic slides and Pappenheim stained. Representative images of three independent biological replicates at 100x magnification.

Article Snippet: Human B—ALL cell lines SEM, RS4;11, REH, and NALM-6 were purchased from DSMZ (Braunschweig, Germany) and maintained at 37 °C and 5% CO 2 in IMDM medium (SEM), Alpha MEM medium (RS4;11) or RPMI 1640 medium (REH, NALM-6), all supplemented with 10% heat-inactivated fetal calf serum and 100 μg/ml penicillin/streptomycin (all PAN—biotech, Aidenbach, Germany).

Techniques: Incubation, Activity Assay, Staining, WST-1 Assay, Calcein AM Assay, Positive Control, Control

Journal: Cell Death Discovery

Article Title: Effective tumor cell abrogation via Venetoclax-mediated BCL-2 inhibition in KMT2A -rearranged acute B-lymphoblastic leukemia

doi: 10.1038/s41420-022-01093-3

Figure Lengend Snippet: A Cells were incubated with increasing concentrations of VEN for 72 h before staining with Annexin V-FITC and propidium iodide and subsequent flow cytometry. Mean ± SD of 1–4 biological replicates; two-way ANOVA with post hoc Dunnett’s multiple comparisons test. Asterisks indicate significance compared to the respective DMSO control. B Dose-response curves (nonlinear regression, curve fit) of cell lines incubated with increasing concentrations of VEN for 72 h. Apoptotic cells were assessed by AnnV/PI staining and flow cytometry and early and late apoptotic/necrotic) cells were added to calculate the amount of apoptotic cells. Mean ± SD of 1–4 biological replicates. C Protein expression of cleaved caspase-3 was analyzed by intracellular flow cytometry after 48 h incubation with 2.5 nM (RS4;11) or 10 nM (SEM) VEN or DMSO (control). Mean ± SD of three biological replicates; paired t -test.

Article Snippet: Human B—ALL cell lines SEM, RS4;11, REH, and NALM-6 were purchased from DSMZ (Braunschweig, Germany) and maintained at 37 °C and 5% CO 2 in IMDM medium (SEM), Alpha MEM medium (RS4;11) or RPMI 1640 medium (REH, NALM-6), all supplemented with 10% heat-inactivated fetal calf serum and 100 μg/ml penicillin/streptomycin (all PAN—biotech, Aidenbach, Germany).

Techniques: Incubation, Staining, Flow Cytometry, Control, Expressing

Journal: Cell Death Discovery

Article Title: Effective tumor cell abrogation via Venetoclax-mediated BCL-2 inhibition in KMT2A -rearranged acute B-lymphoblastic leukemia

doi: 10.1038/s41420-022-01093-3

Figure Lengend Snippet: A Expression of BCL-2 pathway proteins was assessed by immunoblot. Two to three individual biological replicates (gray) and expression mean (red); multiple t -tests, asterisks indicate significance vs time-matched DMSO control. B Total BCL-2 pathway member protein expression was analyzed by intracellular flow cytometry after 48 h incubation. Mean ± SD of three to five biological replicates; ratio paired t -test. C The time-dependent influence of VEN on BCL-2 phosphorylation was measured by immunoblot. BCL-2 and p-BCL-2 bands of three (SEM) or two (RS4;11) individual biological replicates were quantified. Relative BCL-2 phosphorylation values of all replicates (gray), as well as the mean of those experiments (red), are indicated in the graphs. Ratio paired t -test. D Protein expression of phosphorylated and total BCL-2 was assessed by intracellular flow cytometry after 48 h incubation with VEN or DMSO (control). Absolute expression values of controls was set to 100% and the relative change in protein expression following VEN incubation is indicated in the figure. Mean ± SD of five (SEM) or four (RS4;11) biological replicates; paired t -test of each protein vs. respective control. E Functional assessment of Bax-mediated apoptosis induction was performed by Bax translocation assay after 48 h VEN incubation. Four representative images of four biological replicates per cell line and treatment group, 40-fold magnification.

Article Snippet: Human B—ALL cell lines SEM, RS4;11, REH, and NALM-6 were purchased from DSMZ (Braunschweig, Germany) and maintained at 37 °C and 5% CO 2 in IMDM medium (SEM), Alpha MEM medium (RS4;11) or RPMI 1640 medium (REH, NALM-6), all supplemented with 10% heat-inactivated fetal calf serum and 100 μg/ml penicillin/streptomycin (all PAN—biotech, Aidenbach, Germany).

Techniques: Expressing, Western Blot, Control, Flow Cytometry, Incubation, Phospho-proteomics, Functional Assay, Translocation Assay

Journal: Cell Death Discovery

Article Title: Effective tumor cell abrogation via Venetoclax-mediated BCL-2 inhibition in KMT2A -rearranged acute B-lymphoblastic leukemia

doi: 10.1038/s41420-022-01093-3

Figure Lengend Snippet: A Weight progression of ten animals per study group. The dotted lines indicate the treatment period. Multiple t -tests. B , C Tumor cell proliferation was monitored by peripheral blood (PB) blast frequency measurement via flow cytometry (GFP + cells, B ) and in vivo bioluminescence imaging (BLI, C ). Each line represents an individual animal. BLI imaging was discontinued when technical saturation was reached. Mean ± SD of ten animals per group where four animals were sacrificed following therapy finalization at d25 (dotted line); Mann–Whitney test vs. time-matched controls. D Representative BLI images of three individual mice per group in ventral position. E Kaplan–Meier survival analysis. The dotted lines indicate the treatment period. Six animals per group; log-rank test. F The growth rate of leukemic blasts was calculated based on PB blast frequency values measured by flow cytometry. Values of d14 and d21 were used to assess the doubling time during treatment while values of d28 and d35 (SEM) or d35 and d42 (RS4;11) were used for posttreatment calculation. Mean ± SD of three to ten animals per group and time point; Mann–Whitney test. G Pharmacokinetic analyses were conducted one and 2 h after VEN p.o. application to investigate VEN concentrations in PB by liquid coupled mass spectrometry. Each dot represents an individual animal. Mean ± SD of six VEN-treated animals; Wilcoxon matched-pairs signed-rank test. H Analysis of VEN concentration in peripheral blood 24 h after application and the tumor cell doubling time of the respective animal during VEN treatment. Cumulative analysis of SEM and RS4;11-derived xenograft models. Linear regression where each dot represents an individual animal. Pearson’s correlation. I , J Determination of blast frequency in PB, bone marrow (BM), and spleen by flow cytometry ( I ) and spleen parameters ( J ) were assessed when the mice reached humane endpoints (30% blasts in PB or weak performance status). Mean ± SD of five to six animals per group; Mann–Whitney test. K Isolated BM or spleen cells were spun onto microscopic slides and Pappenheim stained. Four representative images of five to six mice per group at 100x magnification.

Article Snippet: Human B—ALL cell lines SEM, RS4;11, REH, and NALM-6 were purchased from DSMZ (Braunschweig, Germany) and maintained at 37 °C and 5% CO 2 in IMDM medium (SEM), Alpha MEM medium (RS4;11) or RPMI 1640 medium (REH, NALM-6), all supplemented with 10% heat-inactivated fetal calf serum and 100 μg/ml penicillin/streptomycin (all PAN—biotech, Aidenbach, Germany).

Techniques: Flow Cytometry, In Vivo, Imaging, MANN-WHITNEY, Mass Spectrometry, Concentration Assay, Derivative Assay, Isolation, Staining

Journal: Leukemia

Article Title: AMPK inhibition enhances apoptosis in MLL-rearranged pediatric B-acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2012.338

Figure Lengend Snippet: Figure 1. AMPK inhibition induces a decrease in cell proliferation and survival. (a) AMPK pathway activation status in BCP-ALL cell lines. MHH-CALL-2 and MHH-CALL-4 are non-translocated cell lines, RS4;11, SEM and ALL-PO carry the (4;11) MLL-AF4 translocation. (b) Cell proliferation rates were determined through MTT assay after treatment with compound C at different times and concentrations. Here the 48 h are shown. DMSO-treated cells viability was set to 100%. GI50 ¼ compound concentration required to inhibit cell proliferation by 50%. (c) Cell viability was determined by flow cytometry with Annexin V–PI staining after treatment with compound C at different times and concentrations. Here the 10 mM 48 h results are shown. DMSO-treated cells viability was set to 100%. Results represent the mean of three independent experiments ±s.e.m. LC50 ¼ compound concentration required to induce cell mortality by 50%. eNOS, endothelial nitric oxide synthase.

Article Snippet: Cell lines and culture Human leukemia cell lines SEM, RS4;11, MHH-CALL-2 and MHH-CALL-4 were purchased from DSMZ German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).

Techniques: Inhibition, Activation Assay, Translocation Assay, MTT Assay, Concentration Assay, Cytometry, Staining

Journal: Leukemia

Article Title: AMPK inhibition enhances apoptosis in MLL-rearranged pediatric B-acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2012.338

Figure Lengend Snippet: Figure 2. AMPK-specific silencing brings to the same effects of compound C treatment. (a) Inhibition of AMPKa1 mRNA expression in SEM, ALL-PO and MHH-CALL-2 cells with both shAMPKalpha1 1 and 2. AMPKa1 mRNA expression in control cells (transfected with un-relevant shRNA sequence) was set to 100%. Results represent mean±s.e.m. of three experiments. mRNA expression levels were measured through Sybr Green real-time quantitative PCR. (b) Cell viability after AMPKa1 silencing in SEM, ALL-PO and MHH-CALL-2 cells was evaluated through Annexin V–PI staining. Control cells viability was set to 100%. Results represent mean±s.e.m. of three experiments. Silenced MLL-rearranged SEM and ALL-PO cells undergo more apoptosis than non-translocated MHH-CALL-2 cells (**Po0.01, ***Po0.001). (c) Inhibition of AMPKa1 protein levels and decrease in the downstream AMPK pathway activation was examined using western blot in control and silenced SEM, ALL-PO and MHH-CALL-2 cells. Representative results are shown with densitometric analysis results indicated under gel images.

Article Snippet: Cell lines and culture Human leukemia cell lines SEM, RS4;11, MHH-CALL-2 and MHH-CALL-4 were purchased from DSMZ German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).

Techniques: Inhibition, Expressing, Control, Transfection, shRNA, Sequencing, SYBR Green Assay, Real-time Polymerase Chain Reaction, Staining, Activation Assay, Western Blot

Journal: Leukemia

Article Title: AMPK inhibition enhances apoptosis in MLL-rearranged pediatric B-acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2012.338

Figure Lengend Snippet: Figure 3. Cell cycle alterations after AMPK inhibition. Results are shown in (a) for SEM cells, (b) for RS4;11 cells and (c) for ALL-PO cells. DMSO and CC (8 mM, 24 h) samples were analyzed by flow cytometry and cell cycle analyses were performed using Multicycle Wincycle software. Representative histograms are shown. The percentage of each phase of the cell cycle (G1, S, G2/M) was calculated. Results come from three independent experiments.

Article Snippet: Cell lines and culture Human leukemia cell lines SEM, RS4;11, MHH-CALL-2 and MHH-CALL-4 were purchased from DSMZ German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).

Techniques: Inhibition, Cytometry, Software

Journal: Leukemia

Article Title: AMPK inhibition enhances apoptosis in MLL-rearranged pediatric B-acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2012.338

Figure Lengend Snippet: Figure 4. Compound C (CC)-mediated cell death follows the mitochondrial pathway (I). Results are shown in (a) for SEM cells, (b) for RS4;11 cells and (c) for ALL-PO cells. In the left panels, depolarization of the mitochondrial transmembrane potential, monitored by the fluorescent dye JC-1, in MLL-rearranged cells treated with CC C (8 mM, 48 h) is reported. The method is based on the ability of this fluorescent probe to enter selectively into the mitochondria, and its color changes reversibly from green to orange as membrane potential increases. This property is due to the reversible formation of JC-1 aggregates on membrane polarization. Aggregation causes a shift in the emitted light from 530 nm (emission by JC-1 monomers) to 590 nm (emission by JC-1 aggregates) following excitation at 490 nm. In middle panels, shown are the mitochondrial production of reactive oxygen species in MLL-rearranged cells treated with CC (8 mM, 48 h). The fluorescence indicator hydroethidine (HE), that is oxidized by superoxide anion into ethidium ion, which emits red fluorescence, was measured. Gray: DMSO-treated cells, red: CC-treated cells. In the right panels, shown are the cleavage of caspase-3 measured by flow cytometry in MLL-rearranged cells after CC (8 mM, 48 h) treatment. Gray: DMSO-treated cells, blue: CC-treated cells.

Article Snippet: Cell lines and culture Human leukemia cell lines SEM, RS4;11, MHH-CALL-2 and MHH-CALL-4 were purchased from DSMZ German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).

Techniques: Membrane, Cytometry