Journal: Blood Cancer Journal

Article Title: Epstein-Barr nuclear antigen 1 induces expression of the cellular microRNA hsa-miR-127 and impairing B-cell differentiation in EBV-infected memory B cells. New insights into the pathogenesis of Burkitt lymphoma

doi: 10.1038/bcj.2012.29

Figure Lengend Snippet: Expression of hsa-miR-127 after transient transfection with EBNA1 in Ramos and human memory B cells. ( a ) The percentage of cells expressing GFP was determined by FACS analysis in Ramos cells 48 h post transfection (Ramos+pmax GFP ) and compared with the control (Ramos mock). A transfection efficiency of 35% was achieved. ( b ) EBNA1 protein levels were evaluated by WB in the EBV-negative BL cell line (Ramos) after transient transfection of EBNA1 . EBNA1 was upregulated 48 h after transfection. Lane 1—Ramos mock; lane 2—Ramos transfected with the empty vector pcDNA3; lane 3—Ramos transfected with pcDNA3- EBNA1 ; lane 4—positive control (Raji). ( c ) Expression of hsa-miR-127 was evaluated by qRT-PCR in the EBV-negative BL cell line (Ramos) after transient transfection of EBNA1 . Hsa-miR-127 was upregulated 48 h after transfection in Ramos cells transfected with EBNA1 , in comparison with the controls (Ramos mock and Ramos+ empty vector). The graph shows the results of three independent experiments. Error bars represent s.d. between replicates. ( d ) The percentage of cells expressing GFP was determined by FACS analysis in memory B cells 24 h post transfection (memory B cells+pmax GFP ) and compared with the control (memory B cells mock). A transfection efficiency of 33% was achieved. ( e ) EBNA1 protein levels in memory B cells were evaluated by indirect immunofluorescence after transient transfection of EBNA1 . EBNA1 was upregulated 24 h after transfection. ( f ) Expression of hsa-miR-127 was evaluated by qRT-PCR in human memory B cells purified from the peripheral blood of normal donors after transient transfection of EBNA1 . Hsa-miR-127 was upregulated 24 h after transfection in memory B cells transfected with EBNA1 , in comparison with the controls (memory B cells mock and memory B cells+ empty vector). The graph shows the results of three independent experiments. Error bars represent s.d. between replicates.

Article Snippet: Raji (EBV-positive BL-derived cell line), Ramos (EBV-negative BL-derived cell line), 293T (human embryonic kidney cell line) and HeLa (cervical cancer cell line) cells were obtained from the American Type Culture Collection (ATCC, Milan, Italy) (references: #CCL-86, #CRL-1596, #CCL-2, #CRL-11268).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Positive Control, Quantitative RT-PCR, Comparison, Immunofluorescence, Purification

Journal: Leukemia

Article Title: Physiological levels of the PTEN-PI3K-AKT axis activity are required for maintenance of Burkitt lymphoma

doi: 10.1038/s41375-019-0628-0

Figure Lengend Snippet: myrAKT overexpression is toxic for BL cell lines. a – f BL cell lines were transduced with lentiviral plasmids expressing constitutively active myrAKT or empty vector (EV). a Transduced cells were FACS-sorted 4 days post transduction and analysed for expression of total AKT and FOXO1 and pAKT T308 and pFOXO1 T24 levels. TUBB served as loading control. A representative of two independent experiments is shown. b Percentage of transduced cells was measured every 3 days using flow cytometry. First measurement was performed 4 days post transduction and the percentage of GFP + cells was set as 100. Data are shown as mean ± SD ( N ≥ 3). c myrAKT downregulates CXCR4. BL cell lines expressing myrAKT or EV were stained with CXCR4-APC or isotype control 5–7 days post transduction. Dot-plots show percentages of CXCR4 + /GFP + and CXCR4 − /GFP + cells. For histograms, only transduced GFP + cells were included. Data are shown as mean ± SD ( N = 3). d Downregulation of critical GC genes after myrAKT overexpression. Transduced cells were sorted 3 days post transduction and RNA expression levels were analysed by qRT-PCR. qRT-PCR data were quantified by the 2 −ΔΔCT method. Data are shown as mean ± SD ( N = 3). The data were analysed by two-sided T -test. For all genes and cell lines p < 0.05 with exception of BL-41 RAD51AP1 p = 0.135. e MYB expression in sorted GFP + cells was analysed by immunoblot 3 days post transduction with myrAKT or EV. TUBB served as loading control. A representative of two independent experiments is shown. f myrAKT activates NF-κB. BL cell lines expressing myrAKT or EV were FACS-sorted 3 days post transduction and pRELA S536 and total RELA were analysed by immunoblot. TUBB served as loading control. A representative of two independent experiments is shown. g Luciferase reporter assay. Namalwa cells stably expressing a NF-κB-dependent luciferase reporter (3 × κB.luc) were transduced with a vector expressing myrAKT. GFP + cells were sorted 4 days post transduction. Luminescence was measured as described in Supplementary Methods. Data are shown as mean ± SD ( N = 3)

Article Snippet: BL cell lines (Ramos, BL-41, Namalwa, Daudi, Jiyoye, Raji) and DLBCL cell lines (BJAB, SU-DHL-5, WSU-NHL, OCI-Ly1, WSU-DLCL2, Karpas-422, HT, OCI-Ly19, SU-DHL-4, and DoHH2) were purchased from DSMZ, Braunschweig, Germany.

Techniques: Over Expression, Transduction, Expressing, Plasmid Preparation, Control, Flow Cytometry, Staining, RNA Expression, Quantitative RT-PCR, Western Blot, Luciferase, Reporter Assay, Stable Transfection

Journal: Oncotarget

Article Title: Dimeric peroxiredoxins are druggable targets in human Burkitt lymphoma

doi:

Figure Lengend Snippet: A. Cytostatic/cytotoxic effects of SK053 on human BL cell lines and normal germinal center B cells (GC B cells). BL cell lines were incubated with SK053 for 48 h and subjected to a MTT viability assay. The LC 50 was calculated in Graphpad Prism 5 by nonlinear regression dose-response analysis with variable slopes. The SEM was calculated based on two independent experiments. GC B cells isolated from human tonsils ( n = 3) were isolated and cultured as described in Methods. Number of viable cells after 48 h treatment with SK053 was assessed using Muse™ Cell Analyzer (Merck Millipore). LC 50 was calculated in Graphpad Prism 5, as described above for BL cell lines. B. Chemical structure of SK053, its biotinylated derivative SK-bio, and the inactive biotinylated analog devoid of the electrophilic center, SK-in. C. Raji-sub cells were incubated with SK-bio or SK-in for 2 h, lysed, and biotin-labeled proteins were affinity-purified on avidin-coated beads. Total protein was resolved by SDS-PAGE and visualized by silver staining. The arrow indicates the band that was excised and identified by mass spectrometry. D. Tandem mass spectra of the Cys-173-containing peptide, HGEVCPAGWKPDGSDTIKPDVQK. The site of cysteine modification is marked with a star. The upper panel spectrum corresponds to a peptide modified with iodoacetamide (+57.021), with the parent ion m/z 802.731 and a charge 3+. The bottom panel presents the spectrum of a peptide in which cysteine bears an inhibitor (+466.225), with parent ion m/z 704.600 and a charge 4+. E. The same samples as in C. were subjected to immunobloting using antibodies specific to PRDX1 and β-actin (ACTIN).

Article Snippet: Human BL cell lines (RAJI, Ramos, BJAB, Daudi, and Namalwa), B-cell chronic lymphocytic leukemia (Mec-1), DLBCL (DHL-4, DHL-6, Ly4, Ly7, Ly10, Ly1, and U2932) and human embryonic kidney (HEK293T) cells were purchased from ATCC.

Techniques: Incubation, MTT Viability Assay, Isolation, Cell Culture, Labeling, Affinity Purification, Avidin-Biotin Assay, SDS Page, Silver Staining, Mass Spectrometry, Modification, Western Blot

Journal: Oncotarget

Article Title: Dimeric peroxiredoxins are druggable targets in human Burkitt lymphoma

doi:

Figure Lengend Snippet: A. Docking of the SK053 molecule to the binding pocket around Cys173 of the rat PRDX1 model (PDB code 1QQ2), in which all rat-specific amino acids were substituted to human-specific counterparts. Active site Cys residues, Cys52 and Cys173, are indicated in red. Distances between the sp2 carbon and Cys thiols are indicated with dashed black lines. Favorable hydrogen bonds are shown as blue dashed lines. B. HEK293T cells were transfected with a pLenti6-PRDX1-V5 plasmid encoding a V5-tagged, wild-type protein (WT), or protein variant with selected cysteines mutated to alanines. Twenty-four hours after transfection, 41 μM (∼ LC 50 ) SK053 (+) or DMSO (−) was added, and after an additional 24 h, the cells were harvested and analyzed by immunoblotting using anti-V5 and anti-PRDX1 antibodies. Note that on the PRDX1 immunoblot (right panel), the highest molecular weight band corresponds to the V5-tagged PRDX1 homodimer (Homo-D V5), the lowest to the untagged homodimer (Homo-D) and the two middle bands to the heterodimers (Hetero-D). C. Scheme presenting double thioalkylation of the active site Cys residues of dimeric 2-Cys PRDXs, resolving Cys (C R ) and peroxidatic Cys (C P ) with SK053.

Article Snippet: Human BL cell lines (RAJI, Ramos, BJAB, Daudi, and Namalwa), B-cell chronic lymphocytic leukemia (Mec-1), DLBCL (DHL-4, DHL-6, Ly4, Ly7, Ly10, Ly1, and U2932) and human embryonic kidney (HEK293T) cells were purchased from ATCC.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Variant Assay, Western Blot, Molecular Weight

Journal: Neoplasia (New York, N.Y.)

Article Title: Smac mimetics and TRAIL cooperate to induce MLKL-dependent necroptosis in Burkitt's lymphoma cell lines

doi: 10.1016/j.neo.2021.03.003

Figure Lengend Snippet: TBZ combination treatment induces caspase-independent cell death in BL. (A) Cells were treated for 24 h with indicated concentrations of BV6. (B) Cells were treated for 24 h (BL-2: 48 h) with BV6 (DG-75, DAUDI: 2 µM, BL-2, BL-60, Salina, RAJI: 4 µM, BJAB: 5 µM, BL-30, BL-70: 6 µM, Seraphine: 7 µM, RAMOS: 18 µM), TRAIL (BL-30: 5 ng/mL, RAMOS/Seraphine: 10 ng/mL, BL-2, DG-75, BL-60, BL-70, Salina, RAJI, BJAB, DAUDI: 15 ng/mL) and zVAD.fmk (20 µM). Cell death was determined by analysis of PI/Hoechst staining and ImageXpress Micro XLS system. Mean and SEM of 3 independent experiments are shown. (C) Protein expression of RIPK1, RIPK3, MLKL, Caspase-8, cIAP1 and cIAP2 was analyzed by Western blotting. GAPDH served as loading control. The asterisk (*) indicates an unspecific band.

Article Snippet: Human BL cell lines RAMOS, DG-75, RAJI, BJAB and DAUDI were obtained from the German Collection of Microorganisms and Cell cultures (DSMZ, Braunschweig, Germany) and BL-2, BL-30, Seraphine, BL-60, BL-70 and Salina cells were kindly provided by T. Oellerich, Department of Medicine II, Hematology/Oncology, University Hospital Frankfurt, Germany.

Techniques: Staining, Expressing, Western Blot, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Smac mimetics and TRAIL cooperate to induce MLKL-dependent necroptosis in Burkitt's lymphoma cell lines

doi: 10.1016/j.neo.2021.03.003

Figure Lengend Snippet: Smac mimetics induce necroptosis in BL . (A) Cells were treated with BV6 (BL-2: 4 µM, BL-30: 6 µM), AT-406, LCL-161 and Birinapant (2,5 µM), TRAIL (BL-30: 5 ng/mL, BL-2: 15 ng/mL) and zVAD.fmk (20 µM) in the presence and absence of Dabrafenib (BL-30: 5 µM, BL-2: 10 µM), Nec-1s (20 µM) and NSA (1,5 µM) for 24 h (BL-30) or 48 h (BL-2). Cell death was determined by analysis of PI/Hoechst staining and ImageXpress Micro XLS system. (B) Cells were treated for 24 h (BL-30, RAJI, RAMOS, Seraphine, DAUDI) or 48 h (BL-2) with BV6 (BL-2: 4 µM, DAUDI: 5 µM, BL-30: 6 µM, Seraphine: 7 µM, RAJI: 8 µM, RAMOS: 18 µM), TRAIL (BL-30: 5 ng/mL, RAMOS/Seraphine: 10 ng/mL, BL-2, RAJI, DAUDI: 15 ng/mL) and zVAD.fmk (20 µM). mRNA expression of TRAIL-R1 and TRAIL-R2 was analyzed by qRT-PCR and fold change normalized to 28s mRNA is shown. Mean and SEM of 3 independent experiments are shown; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Human BL cell lines RAMOS, DG-75, RAJI, BJAB and DAUDI were obtained from the German Collection of Microorganisms and Cell cultures (DSMZ, Braunschweig, Germany) and BL-2, BL-30, Seraphine, BL-60, BL-70 and Salina cells were kindly provided by T. Oellerich, Department of Medicine II, Hematology/Oncology, University Hospital Frankfurt, Germany.

Techniques: Staining, Expressing, Quantitative RT-PCR

Journal: eLife

Article Title: Caspase-mediated cleavage of IRE1 controls apoptotic cell commitment during endoplasmic reticulum stress

doi: 10.7554/eLife.47084

Figure Lengend Snippet:

Article Snippet: Cell line (human) , Ramos , Genentech , RRID: CVCL_0597 , Burkitt’s lymphoma.

Techniques: Knock-Out, Isolation, Clone Assay, CRISPR, Transfection, Construct, FLAG-tag, shRNA, Recombinant, Caspase-Glo Assay, Cell Viability Assay, Flow Cytometry, Fractionation, Cell Culture, Purification, Software