Journal: Molecular cell

Article Title: An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells

doi: 10.1016/j.molcel.2019.05.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Biotinylated lectins (Vector Laboratories, Burlingame, CA) were incubated at different concentrations for 1 h, followed by washing and incubation with Streptavidin-Alexa Fluor488 (Invitrogen) for 1 h. Recombinant human Siglec Fc-chimera (R&D systems, Minneapolis, MN) were precomplexed with Alexa Fluor647 goat anti-human IgG antibody (Life Technologies) for 1 h followed by incubation with cells for 1 hr.

Techniques: Recombinant, Plasmid Preparation, Construct, Software

Journal: Clinical & translational immunology

Article Title: Siglec-7 and Siglec-9 expression in primary triple negative and oestrogen receptor positive breast cancer and in vitro signalling.

doi: 10.1002/cti2.1524

Figure Lengend Snippet: Figure 1. Siglec-7 and Siglec-9 expression is higher in TN tumors than ER+ tumors. (a) Expression of SIGLEC7 and SIGLEC9 from TN (n = 141) and ER+ (n = 808) breast cancer samples was retrieved from the TCGA breast cancer cohort using the UCSC Xena platform. A Mann–Whitney U-test was performed to assess significance (P < 0.01**, P < 0.0001****). (b) Extent of Siglec-7 and Siglec-9 expression in 9 TN and 8 ER+ breast tumors was scored by two researchers. ‘’ indicates negative, ‘+’ slightly positive, ‘++’ positive and ‘+++’ highly positive. (c) TN tumors and (d) ER+ tumors were stained for EpCAM, CD45, Siglec-7, Siglec-9, mIgG1 (isotype control for EpCAM, CD45 and Siglec-7) and mIgG2a (isotype control for Siglec-9). Stainings of 3 representative TN tumors and ER+ tumors are shown. The scale bar represents 100 lm.

Article Snippet: Flow cytometric analysis of Siglec ligands To determine Siglec-7 and Siglec-9 ligand expression on cell lines, a recombinant Siglec-7 Fc, Siglec-9 Fc or IgG1 Fc staining was performed as previously described.32 Briefly, cells were stained with a viability dye, recombinant Siglec Fc proteins (Siglec-7 Fc: 1138-SL-050, Siglec-9 Fc: 1139-SL-050, and IgG1 Fc: 110-HG-100, R&D Systems, Minneapolis, USA) were pre-complexed with Alexa Fluor 488-conjugated goat anti-human IgG (H + L) (A-11013, ThermoFisher Scientific, Waltham, USA) in Hank’s buffered salt solution (14-025-092, ThermoFisher Scientific, Waltham, USA) and subsequently used to stain the cells.

Techniques: Expressing, MANN-WHITNEY, Staining, Control

Journal: Clinical & translational immunology

Article Title: Siglec-7 and Siglec-9 expression in primary triple negative and oestrogen receptor positive breast cancer and in vitro signalling.

doi: 10.1002/cti2.1524

Figure Lengend Snippet: Figure 2. Siglec-7 is expressed on CD3+ cells, CD56+ cells and CD11b+ cells, and Siglec-9 is expressed on CD11b+ cells. Double fluorescent stainings for Siglec-7 or Siglec-9 with either CD3, CD11b, or CD56 and respective isotype controls were performed on breast cancer sections. Panels indicate the following combined markers on one tumor section: (a) Siglec-7 and CD3, (b) Siglec-9 and CD3, (c) Siglec-7 and CD11b, (d) Siglec-9 and CD11b, (e) Siglec-7 and CD56, (f) Siglec-9 and CD56 and (g) isotype controls. Data from a representative TN tumor are shown. The ‘P’ and arrow indicate punctuated Siglec-7 or -9. (a–g) Represent different regions within the tumor section, so only the four panels within one subfigure can be compared. The scale bar indicates 20 lm.

Article Snippet: Flow cytometric analysis of Siglec ligands To determine Siglec-7 and Siglec-9 ligand expression on cell lines, a recombinant Siglec-7 Fc, Siglec-9 Fc or IgG1 Fc staining was performed as previously described.32 Briefly, cells were stained with a viability dye, recombinant Siglec Fc proteins (Siglec-7 Fc: 1138-SL-050, Siglec-9 Fc: 1139-SL-050, and IgG1 Fc: 110-HG-100, R&D Systems, Minneapolis, USA) were pre-complexed with Alexa Fluor 488-conjugated goat anti-human IgG (H + L) (A-11013, ThermoFisher Scientific, Waltham, USA) in Hank’s buffered salt solution (14-025-092, ThermoFisher Scientific, Waltham, USA) and subsequently used to stain the cells.

Techniques:

Journal: Clinical & translational immunology

Article Title: Siglec-7 and Siglec-9 expression in primary triple negative and oestrogen receptor positive breast cancer and in vitro signalling.

doi: 10.1002/cti2.1524

Figure Lengend Snippet: Figure 3. TN and ER+ tumors express high levels of Siglec-7 ligands and Siglec-9 ligands. Siglec-7 and Siglec-9 ligands were stained using biotinylated Siglec-7 or Siglec-9 in (a) TN tumors and (b) ER+ tumors. Sections were additionally stained for EpCAM, CD45 and their isotype control mIgG1. For each subtype, a representative tumor is shown. As control for Siglec ligands, the sections were stained with streptavidin-HRP. ‘T’ indicates tumor cells and ‘S’ indicates stroma. The scale bar equals 20 lm.

Article Snippet: Flow cytometric analysis of Siglec ligands To determine Siglec-7 and Siglec-9 ligand expression on cell lines, a recombinant Siglec-7 Fc, Siglec-9 Fc or IgG1 Fc staining was performed as previously described.32 Briefly, cells were stained with a viability dye, recombinant Siglec Fc proteins (Siglec-7 Fc: 1138-SL-050, Siglec-9 Fc: 1139-SL-050, and IgG1 Fc: 110-HG-100, R&D Systems, Minneapolis, USA) were pre-complexed with Alexa Fluor 488-conjugated goat anti-human IgG (H + L) (A-11013, ThermoFisher Scientific, Waltham, USA) in Hank’s buffered salt solution (14-025-092, ThermoFisher Scientific, Waltham, USA) and subsequently used to stain the cells.

Techniques: Staining, Control

Journal: Clinical & translational immunology

Article Title: Siglec-7 and Siglec-9 expression in primary triple negative and oestrogen receptor positive breast cancer and in vitro signalling.

doi: 10.1002/cti2.1524

Figure Lengend Snippet: Figure 4. chSiglec-7 signalling is induced by multiple TN cell lines and one ER+ tumor cell line. (a) Siglec-7 Fc and Siglec-9 Fc ligands on SiaFEt- or DMSO-treated breast cancer cell lines were determined using flow cytometry, using IgG1 Fc as isotype control. Cell lines were treated for 3 days with 31.5 lM SiaFEt or DMSO as vehicle control. Representative data of n = 2 or 3 experiments are shown. (b) chSiglec-7 cells were validated for functionality by culture with PMA/ionomycin, by culture on wells coated with anti-Siglec-7 antibody or its respective mIgG1 isotype control or by culture with sialic acid polymer (left panel). 2 Technical replicates per condition are shown of a representative luciferase assay. chSiglec-7 Jurkat/MA cells were co-cultured with SiaFEt- or DMSO-treated tumor cell lines and subsequently chSiglec-7 signalling was assessed by luciferase assays (right panel). Luciferase assays were repeated 3 times, each containing 2–3 technical replicates per condition. Luminescence values are normalised to those of unstimulated chSiglec-7. Bars represent mean SD of 3 luciferase assays. Matched one-way ANOVAs were performed on log-transformed raw data (adjusted P < 0.05*, P < 0.01**). (c) Correlation between Siglec-7 ligand expression and chSiglec-7 induction by the cell lines. Luminescence values of chSig7 co-culture with DMSO-treated tumor cells are normalised to those of co-culture with SiaFEt-treated tumor cells. Dots represent mean SD of 3 luciferase assays.

Article Snippet: Flow cytometric analysis of Siglec ligands To determine Siglec-7 and Siglec-9 ligand expression on cell lines, a recombinant Siglec-7 Fc, Siglec-9 Fc or IgG1 Fc staining was performed as previously described.32 Briefly, cells were stained with a viability dye, recombinant Siglec Fc proteins (Siglec-7 Fc: 1138-SL-050, Siglec-9 Fc: 1139-SL-050, and IgG1 Fc: 110-HG-100, R&D Systems, Minneapolis, USA) were pre-complexed with Alexa Fluor 488-conjugated goat anti-human IgG (H + L) (A-11013, ThermoFisher Scientific, Waltham, USA) in Hank’s buffered salt solution (14-025-092, ThermoFisher Scientific, Waltham, USA) and subsequently used to stain the cells.

Techniques: Cytometry, Control, Polymer, Luciferase, Cell Culture, Transformation Assay, Expressing, Co-Culture Assay

Journal: Journal of translational medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics.

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Fig. 5 Observation of CD19-BBζ-CAR expression in relapsed Nalm-6 cells and salvage treatment. A Detection of FMC63 and CD247 transcripts and 4-1BB gene of CAR in CD19+ Nalm-6 (red) and relapsed CD19− Nalm-6 cells (blue) by qRT-PCR. Data of left bar graph represent the relative quantification using ACTB as the internal reference. Error bars represent s.d. The data are the representative of three independent experiments. B Expression of CD19 and CAR on CD19+ Nalm-6 cells and relapsed CD19− Nalm-6 cells analyzed by flow cytometry (representative of 3 experiments). Merge Graphs, the blue dots represent CD19− Nalm-6 cells and the red dots represent Nalm-6 cells. C Confocal imaging of Nalm-6 cells and relapsed CD19− Nalm-6 cells using Alexa Flour 488-conjugated anti-CD19 antibody (green), Alexa Flour 647-conjugated anti-CAR19 antibody (red), and DAPI (blue). D Lentiviral integration sites of CAR transduced Nalm-6 cells were analyzed by linear-amplification mediated PCR (LAM-PCR) and visualized with Circos plots. The integration sites across the genome and genomic features were shown from outer to inner circle: (1) cytogenetic bands; (2) genes that harbor these integration sites along with a bar chart showing the reads of integration sites; (3) the distribution of integration sites, with colored circles representing different gene functional regions of the host sequence: purple for promoter region, green for intron region, and red for distal intergenic region. E Phenotype changes of Nalm-6 cells transduced with small amount of CD19 CAR lentiviruses detected by flow cytometry over time. Gating was based on the same cells stained with isotype-matched antibody. F Dynamics of CD19− B phenotype in relapsed cells after co-culture with different ratios (5×, 20×) of Nalm-6 cells. Gating was based on the same cells stained with isotype-matched antibody. G Relapsed CD19− Nalm-6 cells were tested by qPCR specific for VSV-G sequence. H Comparison of in vitro efficacy of CD19-, CD22-, CD19/CD22- and CD22×CD19- CAR T cells. Cocultures with the relapsed cells were performed at 1:5, 1:1, and 5:1 E: T ratios, and lysis efficacies were detected by the LDH release assay Declarations

Article Snippet: The cells were then washed twice and stained with phycoerythrin (PE) streptavidin (BD bioscience, USA) for 15 min. CART-22 cells and CART-22/19 cells were washed once and incubated with CD22 Fc Alexa Fluor® 647 Protein (R&D Systems, USA) for 15 min. To detect in vitro cytotoxicity of CART-19 cells, transduced and untransduced T cells were co-cultured with Nalm-6 cells (total 1 × 106 cells) at E: T ratios (0.2:1, 0.5:1, 1:1, 5:1) for 6, 24 and 72 h. Cells were pipetted to incubate with antibodies for 30 min at room temperature in the dark and washed twice with PBS.

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Flow Cytometry, Imaging, Amplification, Functional Assay, Sequencing, Transduction, Staining, Co-Culture Assay, Comparison, In Vitro, Lysis, Lactate Dehydrogenase Assay