Journal: Cancer Medicine

Article Title: SNGH16 regulates cell autophagy to promote Sorafenib Resistance through suppressing miR‐23b‐3p via sponging EGR1 in hepatocellular carcinoma

doi: 10.1002/cam4.3020

Figure Lengend Snippet: The lncRNA microarray data from Hep3B and Hep3B/So cells

Article Snippet: Human LncRNA Microaray V1.0 (Arraystar) was used to analyze the data of five replicate samples, calculate the p ‐value of the gene expression difference and statistical significance, and use the Fold Change (FC) ≥1.5 or ≤−1.5, P < .05 for t test as a criterion for differential gene screening.

Techniques: Microarray

Journal: Frontiers in Immunology

Article Title: Applications of Protein Microarrays in Biomarker Discovery for Autoimmune Diseases

doi: 10.3389/fimmu.2021.645632

Figure Lengend Snippet: Major protein microarray platforms for biomarker discovery in autoimmune diseases.

Article Snippet: The HuProtTM Human Proteome Microarray V1.0 was designed by Zhu lab at Johns Hopkins School of Medicine and produced by CDI Laboratories around 2012, containing 16,368 recombinant GST-His-tagged human proteins expressed in S. cerevisiae , and the current v4.0 contains more than 21,000 unique proteins.

Techniques: Microarray, Biomarker Discovery, Protein Array, Recombinant, In Silico, Selection, In Situ, Peptide Microarray, Ab Array, Bioprocessing

Journal: Frontiers in Immunology

Article Title: Applications of Protein Microarrays in Biomarker Discovery for Autoimmune Diseases

doi: 10.3389/fimmu.2021.645632

Figure Lengend Snippet: Selective application of protein microarrays in biomarker discovery for autoimmune diseases.

Article Snippet: The HuProtTM Human Proteome Microarray V1.0 was designed by Zhu lab at Johns Hopkins School of Medicine and produced by CDI Laboratories around 2012, containing 16,368 recombinant GST-His-tagged human proteins expressed in S. cerevisiae , and the current v4.0 contains more than 21,000 unique proteins.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Western Blot, Microarray, Immunopeptidomics, Protein Array, Diagnostic Assay, Recombinant, Purification, Derivative Assay, Competitive ELISA, Peptide Microarray, Software, Construct, Bioprocessing, Ab Array, Clinical Proteomics, Expressing, Amplified Luminescent Proximity Homogenous Assay, Multiplex Assay, Glycoproteomics, Membrane, Sequencing, Immunofluorescence, Staining, Virus, Immunohistochemistry, cDNA Library Assay, Clone Assay, Suspension, Apoptosis Assay, Stripping Membranes, In Silico, Activity Assay, Cytokine Assay

Journal: Cell Reports Medicine

Article Title: Identification and targeting of protein tyrosine kinase 7 (PTK7) as an immunotherapy candidate for neuroblastoma

doi: 10.1016/j.xcrm.2023.101091

Figure Lengend Snippet: Design, expression, and specificity of UBC/EF1a-based PTK7 CARs (A) Lentiviral vector-based UBC PTK7 CAR DNA constructs. (B) Percentage GFP + Jurkat T cells transduced with PTK7 VL-L-VH CAR or PTK7 VH-L-VL CAR, gated on live cells, n = 3 biological replicates. (C) Percentage CAR+ (PTK7-FC chimera+) Jurkat T cells transduced with PTK7 VL-L-VH CAR or PTK7 VH-L-VL CAR. Gated on live GFP + cells, n = 3 biological replicates. (D) Representative flow plot showing GFP vs. CAR expression in Jurkat T cells, gated on live cells. (E) CAR western blot from whole-cell lysate of Jurkat T cells transduced with PTK7 VL-L-VH CAR or PTK7 VH-L-VL CAR, 3 days post-transduction. Western blot antibody against human CD3ζ. (F) Percentage CD69 + Jurkat T cells co-cultured with (+) or without (-) target cells, gated on live GFP + cells, n = 3 biological replicates. (G) Top: PTK7 and GAPDH western blot of whole-cell lysate of Jurkat T cells and primary T cells. Bottom: histogram depicting cell surface PTK7 expression on T cells pre- and post-stimulation with CD3/CD28 beads relative isotype control. (H) Left: percentage CD69 + Jurkat T cells transduced with PTK7 VH-L-VL CAR or CD19 CAR co-cultured with target cells (SK-N-AS non-targeting gRNA control or SK-N-AS PTK7 KO), gated on live GFP + cells, n = 3 biological replicates. Right: representative flow plots showing CD69 expression within GFP + Jurkat T cells. (I) Lentiviral vector-based EF1α PTK7 CAR DNA construct. (J) Left: representative flow plot showing CAR expression (PTK7-FC chimera) in primary T cells transduced with indicated CAR AT MOI 20. Right: MFI of PTK7 CAR expression in primary T cells transduced with indicated CAR, n = 2 or 3 biological replicates. (K) Western blot from whole-cell lysate of primary T cells transduced with indicated CAR at MOI 20. Western blot antibody against human CD3ζ.

Article Snippet: Cells were then washed with FACS buffer and stained with 3 μL anti-CD69-APC-Cy7 antibody (BD Pharmingen), washed 1x with FACS buffer, and detected by flow cytometry (Aurora Cytek) and analyzed by FlowJo software (v10).

Techniques: Expressing, Plasmid Preparation, Construct, Transduction, Western Blot, Cell Culture

Journal: Cell Reports Medicine

Article Title: Identification and targeting of protein tyrosine kinase 7 (PTK7) as an immunotherapy candidate for neuroblastoma

doi: 10.1016/j.xcrm.2023.101091

Figure Lengend Snippet: PTK7 CAR T cells have specific in vitro cytotoxicity (A) Twelve-hour flow cytometry cytotoxicity assay. Percentage cytotoxicity = the sum of 7AAD + , annexin V + , and 7AAD + annexin V+ cells, gated on target cells only. n = 3 donors with n = 2 biological replicates. Four-hour cytotoxicity shown in Figure S5 A. (B) Target cells and CAR T cells or mock T cell control were incubated with caspase-3/7 dye and live cell analysis was performed at the indicated time points. Processing definitions of caspase-3/7 activity (green object count) on target cells were determined. n = 1 donor. Error bars represent SD of 4 technical replicates. (C) Percentage CD69 expression in mock T cells or CAR T cells co-cultured with indicated target cells from 4-h cytotoxicity assay, gated on live primary T cells. n = 3 donors, statistical analysis represents Student’s t test (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns, p > 0.05). (D) PTK7 MFI expression in MR5, NLF, and CMK target cells, n = 6 biological replicates. (E) Representative image of mock or CAR T cells with target cell co-culture at 4 h. Scale bar provided is applicable to each image in E.

Article Snippet: Cells were then washed with FACS buffer and stained with 3 μL anti-CD69-APC-Cy7 antibody (BD Pharmingen), washed 1x with FACS buffer, and detected by flow cytometry (Aurora Cytek) and analyzed by FlowJo software (v10).

Techniques: In Vitro, Flow Cytometry, Cytotoxicity Assay, Incubation, Activity Assay, Expressing, Cell Culture, Co-Culture Assay

Journal: Cell Reports Medicine

Article Title: Identification and targeting of protein tyrosine kinase 7 (PTK7) as an immunotherapy candidate for neuroblastoma

doi: 10.1016/j.xcrm.2023.101091

Figure Lengend Snippet:

Article Snippet: Cells were then washed with FACS buffer and stained with 3 μL anti-CD69-APC-Cy7 antibody (BD Pharmingen), washed 1x with FACS buffer, and detected by flow cytometry (Aurora Cytek) and analyzed by FlowJo software (v10).

Techniques: Microarray, Recombinant, Cell Isolation, Mass Spectrometry, CRISPR, Negative Control, Software, Lysis, Protease Inhibitor, Bradford Protein Assay, BIA-KA, Binding Assay