Journal: PLoS ONE

Article Title: Translational Regulation of GPx-1 and GPx-4 by the mTOR Pathway

doi: 10.1371/journal.pone.0093472

Figure Lengend Snippet: The effect of imatinib on GPx enzyme activity (A) and GPx-1 protein (B) in MEG-01, MDA-MB-231 and GM10832 is shown. GPx activity was increased 2-fold, and protein increased 4-fold ( P <0.01) by 300 nM imatinib treatment in MEG-01. GPx-1 activity and protein levels did not change following imatinib treatment of MDA-MB-231 (500 nM imatinib) and GM10832 (300 nM). Data presented in A is the result of three independent experiments * = P <0.001. Error bars indicate S.D.

Article Snippet: The GM10832 human immortal B lymphocyte cell line was obtained from the National Institute of General Medical Sciences Human Genetic Cell Repository of the Coriell Institute (Camden, NJ).

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Translational Regulation of GPx-1 and GPx-4 by the mTOR Pathway

doi: 10.1371/journal.pone.0093472

Figure Lengend Snippet: The effect of rapamycin on GPx-1 activity (A) and protein levels (B) in KU812a, MEG-01 GM10832 or MDA-MB-231 is shown. The effect of rapamycin on steady-state GPx-1 transcript levels as determined by RT-qPCR and normalization of GPx-1 Ct values to Ct values for 18s RNA (C). Rapamycin significantly increased protein respectively 3-fold and 1.3-fold in KU812a and MEG-01 ( P = 0.05). Steady state transcript levels for GPx-1 were unaffected by treatment with rapamycin in KU812a. Data shown is representative of three independent experiments. * = P <0.001, ** = P <0.01. † = P <0.05. Error bars indicate S.D.

Article Snippet: The GM10832 human immortal B lymphocyte cell line was obtained from the National Institute of General Medical Sciences Human Genetic Cell Repository of the Coriell Institute (Camden, NJ).

Techniques: Activity Assay, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Translational Regulation of GPx-1 and GPx-4 by the mTOR Pathway

doi: 10.1371/journal.pone.0093472

Figure Lengend Snippet: The effect of 1 ng/mL rapamycin on GPx-4 and MnSOD and pS6 protein levels in KU812a, MEG-01, GM10832 and MDA-MB-231 is shown. GPx-4 by 3-day 1 ng/mL rapamycin treatment in KU812a and MEG-01 ( P > 0.2), but was increased 3-fold in GM10832 ( P = 0.05) and 6-fold in MDA-MB-231 cells ( P = 0.02). The disappearance of pS6 signal following rapamycin treatment indicates inhibition of mTOR. Data shown is representative of three independent experiments.

Article Snippet: The GM10832 human immortal B lymphocyte cell line was obtained from the National Institute of General Medical Sciences Human Genetic Cell Repository of the Coriell Institute (Camden, NJ).

Techniques: Inhibition

Journal: PLoS ONE

Article Title: A Novel BAT3 Sequence Generated by Alternative RNA Splicing of Exon 11B Displays Cell Type-Specific Expression and Impacts on Subcellular Localization

doi: 10.1371/journal.pone.0035972

Figure Lengend Snippet: HeLa cells (Adenocarcinoma), the human melanoma cell line MelJuSo, the B lymphoma Raji and monocyte-derived dendritic cells (moDCs) were plated on coverslips and stained for BAT3 using a polyclonal serum against a C-terminal peptide (middle lane). Cell nuclei (left lane) were visualized with DAPI (A) or 7AAD (B). Merged images are shown in the right lane. A. Immunofluorescence staining was evaluated with a standard fluorescence microscope and B. by confocal microscopy. Scale bars = 10 µm. C. Nuclear and cytosolic staining of endogenous BAT3 in Raji cells was evaluated in 10 single cells using ImageJ. MFI, mean fluorescence intensity per region of interest D. Western blot analysis of subcellular fractions from Raji and HeLa cells. Nuclei (N) and cytoplasm (C) were separated by SDS-PAGE and immunoblotted for BAT3, GADPH (cytosolic marker) and histone H3 (nuclear marker).

Article Snippet: The B lymphoma cell line Raji was cultured in RPMI and dendritic cells were grown in VLE-RPMI supplemented with antibiotics and glutamine (Biochrom AG, Berlin, Germany).

Techniques: Derivative Assay, Staining, Immunofluorescence, Fluorescence, Microscopy, Confocal Microscopy, Western Blot, SDS Page, Marker

Journal: PLoS ONE

Article Title: A Novel BAT3 Sequence Generated by Alternative RNA Splicing of Exon 11B Displays Cell Type-Specific Expression and Impacts on Subcellular Localization

doi: 10.1371/journal.pone.0035972

Figure Lengend Snippet: A. HeLa cells transfected with BAT3 Δ11B,24 were cultured on coverslips in the presence (lower panel) or absence (upper panel) of leptomycin B (LMB) for 2 h. Cells were subsequently stained with V5 mAb and inspected with standard immunofluorescence microscopy (second panel). Left panel shows DAPI stained nuclei, third panel merging of images and right panel displays phase contrast images. Scale bars = 10 µm. B. Raji cells were cultured for 2 h in the presence (lower panel) or absence (upper panel) of LMB and then plated on coverslips. Cells were subsequently stained with the polyclonal anti-BAT3 serum and with ISCR3 mAb (HLA-DR) for evaluation by immunofluorescence microscopy. Left panel shows DAPI staining, second panel staining for BAT3, third panel staining for HLA-DR and images were merged in the right panel. Scale bars = 5 µm.

Article Snippet: The B lymphoma cell line Raji was cultured in RPMI and dendritic cells were grown in VLE-RPMI supplemented with antibiotics and glutamine (Biochrom AG, Berlin, Germany).

Techniques: Transfection, Cell Culture, Staining, Immunofluorescence, Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Dual role of the peptide-loading complex as proofreader and limiter of MHC-I presentation

doi: 10.1073/pnas.2321600121

Figure Lengend Snippet: Deletion of PLC components reduces MHC-I surface levels and changes the MHC-I surface composition. MHC-I surface levels of HAP1 cells with single knockouts of PLC components (wt, gray; ΔHLA, red; ΔTAP1, dark blue; ΔTAP2, light blue; ΔTSN, orange; ΔCRT, yellow; ΔERp57, green; ΔERAP1, purple). Flow cytometric analysis of total MHC-I (W6/32), A*02:01 (BB7.2), and B*40:01 (JOAN-1) surface levels was performed by using the respective primary antibody and Nb AF647 . Exemplary histograms and surface quantity of total MHC-I ( A ), A*02:01 ( B ), and B*40:01 ( C ) molecules per cell. The number of MHC-I surface molecules was determined by using Quantum TM AF647 MESF microspheres and normalizing to ΔHLA cells (mean ± SD, n = 4). Proportion of A*02:01 ( D ), B*40:01 ( E ), and the sum of A*02:01 and B*40:01 proportions ( F ) of the total MHC-I molecules (mean ± SD, n = 4). Dark and light colors correspond to A*02:01 and B*40:01 molecules, displayed in ( D ) and ( E ), respectively. The black dashed line represents the value of wt cells. Welch ANOVA comparing ΔTAP1, ΔTAP2, ΔTSN, ΔCRT, ΔERp57, and ΔERAP1 with HAP1 wt cells was performed (ns, nonsignificant; * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: HAP1 wt cell line (HLA allomorphs A*02:01, B*40:01, C*03:04) and HAP1 knockout cell lines (ΔHLA with knockouts of HLA-A, -B, -C, and -G, ΔTAP1, ΔTSN, ΔCRT, ΔERp57, ΔERAP1) were kindly provided by Robbert Spaapen (Sanquin Research, Netherlands) ( ).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Dual role of the peptide-loading complex as proofreader and limiter of MHC-I presentation

doi: 10.1073/pnas.2321600121

Figure Lengend Snippet: Knockout of PLC components increases the ratio of suboptimally loaded A*02:01 surface complexes. Flow cytometric analyses of extracellular peptide exchange on HAP1 wt cells and HAP1 cells with knockouts of individual components of the antigen-processing machinery after pulsing with ELA ( Top ) or TQV ( Bottom ) peptide (1 µM each). ELA and TQV peptide were used crosswise for background correction. ( A ) Activation of the reporter T cells DMF5 NFκB::eGFP ( Top ) and 1G4 NFκB::eGFP ( Bottom ) upon coculture with pulsed HAP1 cells. T cell activation was determined by eGFP median fluorescence intensity (MFI) and normalized to coculture with HAP1 wt cells (mean ± SD, n = 4). ( B ) Number of ELA-A2*02:01 ( Top ) and TQV-A2*02:01 complexes ( Bottom ) per cell was determined by using the enhanced-affinity sTCRs MEL5 Spy-AF647 and 1G4 Spy-AF647 , respectively, and Quantum TM AF647 MESF microspheres (mean ± SD, n = 4). ( C ) Presentation of ELA-A2*02:01 ( Top ) and TQV-A2*02:01 complexes ( Bottom ) in relation to A*02:01 levels and HAP1 wt cells, illustrating the relative peptide exchange (mean ± SD, n = 4). The black dashed line represents the value of HAP1 wt cells. Welch ANOVA comparing ΔTAP1, ΔTAP2, ΔTSN, ΔCRT, ΔERp57, and ΔERAP1 with HAP1 wt cells was performed (ns, nonsignificant; * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: HAP1 wt cell line (HLA allomorphs A*02:01, B*40:01, C*03:04) and HAP1 knockout cell lines (ΔHLA with knockouts of HLA-A, -B, -C, and -G, ΔTAP1, ΔTSN, ΔCRT, ΔERp57, ΔERAP1) were kindly provided by Robbert Spaapen (Sanquin Research, Netherlands) ( ).

Techniques: Knock-Out, Activation Assay, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Dual role of the peptide-loading complex as proofreader and limiter of MHC-I presentation

doi: 10.1073/pnas.2321600121

Figure Lengend Snippet: Deficiencies in the editing module lead to elevated presentation of abundant, high-affinity peptides. Flow cytometric analyses of ELA-A*02:01 and TQV-A*02:01 presentation by HAP1 wt cells and HAP1 cells with knockouts of individual components of the antigen-processing machinery upon transfection with plasmids encoding for peptide expression of either ELA ( Top ) or TQV ( Bottom ). ELA and TQV peptide were used crosswise for background correction. ( A ) Activation of DMF5 NFκB::eGFP ( Top ) and 1G4 NFκB::eGFP ( Bottom ) reporter T cells upon coculture with peptide-expressing HAP1 cells. T cell activation was determined by eGFP MFI and normalized to coculture with HAP1 wt cells (mean ± SD, n = 4). ( B ) Number of ELA-A2*02:01 ( Top ) and TQV-A2*02:01 complexes ( Bottom ) per peptide-expressing cell was determined by using enhanced-affinity sTCRs MEL5 Spy-AF647 and 1G4 Spy-AF647 , respectively, and Quantum TM AF647 MESF microspheres (mean ± SD, n = 4). ( C ) Presentation of ELA-A2*02:01 ( Top ) and TQV-A2*02:01 complexes ( Bottom ) in relation to A*02:01 levels in peptide-expressing HAP1 cells and HAP1 wt cells (mean ± SD, n = 4). The black dashed line represents the value of HAP1 wt cells. Welch ANOVA comparing ΔTAP1, ΔTAP2, ΔTSN, ΔCRT, ΔERp57, and ΔERAP1 with HAP1 wt cells was performed (ns, nonsignificant; * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: HAP1 wt cell line (HLA allomorphs A*02:01, B*40:01, C*03:04) and HAP1 knockout cell lines (ΔHLA with knockouts of HLA-A, -B, -C, and -G, ΔTAP1, ΔTSN, ΔCRT, ΔERp57, ΔERAP1) were kindly provided by Robbert Spaapen (Sanquin Research, Netherlands) ( ).

Techniques: Transfection, Expressing, Activation Assay

Journal: Transplantation and cellular therapy

Article Title: NK Cell Adoptive Immunotherapy of Cancer: Evaluating Recognition Strategies and Overcoming Limitations

doi: 10.1016/j.bbmt.2020.09.030

Figure Lengend Snippet: Sample List of Active Trials Involving Allogeneic NK Cells

Article Snippet: Ospedale Papa Giovanni XXIII Haploidentical cytokine-induced killer cells Relapsed hematologic malignancy after transplant NCT03853317 NantKwest, Inc.; NantCell, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Avelumab N-803 Merkel cell carcinoma NCT03937895 SMT bio Co., Ltd. NK cells Pembrolizumab Advanced biliary tract cancer NCT03940833 Asclepius Technology Company Group (Suzhou) Co., Ltd. NK92 cell line modified to express B cell maturation antigen CAR Multiple myeloma NCT04162158 Beijing 302 Hospital; Shenzhen Third People's Hospital; The First People's Hospital of Zhengzhou NK cells Advanced hepatocellular carcinoma NCT04309084 Celularity Incorporated Human placental CD34 + -derived NK cells Multiple myeloma NCT04310592 Celularity Incorporated Human placental CD34 + -derived NK cells Acute myeloid leukemia Open in a separate window Sample List of Active Trials Involving Allogeneic NK Cells Regardless of the source, it is increasingly evident that allogeneic NK cells are potentially advantageous for adoptive immunotherapy of malignancy, because list-behavior=enumerated prefix-word= mark-type=lower-roman max-label-size=0 they may be used in an off-the-shelf setting, their low association with GVHD, and they do not harbor the same inherent dysfunction as autologous patient-derived NK.

Techniques: Transplantation Assay, Modification, Transfection, Expressing, Derivative Assay