Journal: Advanced healthcare materials

Article Title: Urine-Stable Aptamer-Conjugated Gold Nanorods for the Early Detection of High-Grade Bladder Cancer Residual Disease.

doi: 10.1002/adhm.202403314

Figure Lengend Snippet: Figure 1. Features of the aptamer and tested bladder cancer cell lines. A) The secondary structure of H02 full-length and Apt-Itg (nucleotides from 18 to 51, boxed); structures of the H02 full-length and Apt-Itg were predicted by using Mfold web server, and the calculated change in Gibbs free energy (ΔG) values are reported. B) AlphaFold 3 molecular docking result of integrin 𝛼5𝛽1-Apt-Itg binding complex; Apt-Itg, integrin 𝛼5 and integrin 𝛽1 predicted structures are shown in yellow, light blue and red, respectively. The predicted interaction of Apt-Itg with integrin 𝛼5, involving D154 and Y208 residues, is framed on the side. C) The expression of integrin 𝛼5𝛽1 by human and murine bladder cancer cell lines evaluated by western blot analysis of the 𝛼5 and 𝛽1 chain of the integrin. The expression of the housekeeping protein 𝛼-tubulin was used to estimate the relative fold of expression of integrin chains D). MB49-Luc; murine bladder cancer cell line.

Article Snippet: For cell binding of anti-integrin α5 antibody (Ab-Itg), 2.0 × 105 T24 cells were detached from culture plates as described above and left untreated or incubated with Human Integrin alpha 5/CD49e Alexa Fluor488-conjugated Antibody (dilution 1:10, R&D system, Minneapolis, MN) in DPBS supplemented with 0.1% bovine serum albumin.

Techniques: Binding Assay, Expressing, Western Blot

Journal: Advanced healthcare materials

Article Title: Urine-Stable Aptamer-Conjugated Gold Nanorods for the Early Detection of High-Grade Bladder Cancer Residual Disease.

doi: 10.1002/adhm.202403314

Figure Lengend Snippet: Figure 4. Integrin 𝛼5 silencing results in reduced Apt-Itg binding. A) T24, RT112, and MB49-Luc cells were left untreated (NT) or transfected with the indicated Integrin 𝛼5 siRNAs or siRNA ctrl. At 24 h post-transfection, cells were harvested, and cell lysates prepared and immunoblotted with anti- integrin 𝛼5 or anti-integrin 𝛽1 antibody. Equal loading was confirmed by immunoblot with anti-𝛼-tubulin antibody. The histograms indicate the integrin chains/𝛼-tubulin ratio of the densitometric signals. Values are shown relative to siRNA ctrl, arbitrarily set to 1. B) Binding of 1 μm Alexa647-Apt-Itg to human and murine bladder cancer cells following 24 h-transfection with si-ITG𝛼5 (blue) and siRNA ctrl (red). C) Binding of Alexa488-Ab-Itg to T24 cells 24 h post-transfection. (B, C) The histogram indicates gMFI of aptamer- or antibody-treated cells normalized to the gMFI of siRNA ctrl untreated cells (black), arbitrarily set to 1. Bars depict mean ± SD of two independent experiments. *p < 0.005, **p < 0.001, and ***p < 0.0001.

Article Snippet: For cell binding of anti-integrin α5 antibody (Ab-Itg), 2.0 × 105 T24 cells were detached from culture plates as described above and left untreated or incubated with Human Integrin alpha 5/CD49e Alexa Fluor488-conjugated Antibody (dilution 1:10, R&D system, Minneapolis, MN) in DPBS supplemented with 0.1% bovine serum albumin.

Techniques: Binding Assay, Transfection, Western Blot

Journal: Oncogene

Article Title: C1GALT1 high expression is associated with poor survival of patients with pancreatic ductal adenocarcinoma and promotes cell invasiveness through integrin α v

doi: 10.1038/s41388-020-01594-4

Figure Lengend Snippet: A Western blots showing C1GALT1 stable knockdown in HPAF-II and HPAC cells and C1GALT1 overexpression in MIAPaca2 cells. PDAC cells were transfected using lentivirus-mediated C1GALT1 shRNA in pLKO.1 vector (shC1GALT1) compared with its empty vector (shControl) and C1GALT1 was overexpressed using C1GALT1/pcDNA3.1 plasmid (C1GALT1) compared with its empty plasmid (mock). GAPDH was used as an internal loading control. B C1GALT1 knockdown or overexpression did not alter the expression of surface integrins β 1 , α 5 , α 5 β 1 , and α v in HAPF-II and HPAC cells analyzed by flow cytometry. Unstained cells were used as a negative control (-). C Effects of functional blocking antibodies against integrins on PDAC cell invasion. C1GALT1 knockdown HPAF-II and HPAC cells and C1GALT1 overexpressing MIAPaca2 cells were subjected to Matrigel invasion assays. Cells were treated with 10 μg/mL of blocking antibody, as indicated. IgG was used as a control. Invasion of HPAF-II, HPAC, and MIAPaca2 cells was analyzed after 24 and 48 h. Results are presented as mean ± SD of four independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001 by student’s t test. D Effects of functional blocking antibodies on FAK phosphorylation in PDAC cells using Western blotting. Functional blocking antibody against integrin α v or α 5 was used, as indicated, to treat HPAF-II and HPAC cells for 30 min before seeding to culture plates. IgG was used as a control. GAPDH was used as an internal loading control. E Western blots showing changes in Tn antigens on cell surface integrins α V and α 5 . Cells were surface biotinylated. Plasma Membrane Protein Extraction Kit (Abcam) was used for extraction and purification of plasma membrane proteins from HPAF-II and HPAC cells. VVA pull-down (PD) assays were performed to assess changes in Tn antigens on integrins. Proteins were detected by Western blot (WB) analysis. F A schematic diagram illustrating the proposed mechanism by which C1GALT1 promotes tumor growth and metastasis in pancreatic cancer. C1GALT1 modifies O-glycans on integrins, including α 5 , α v , and β 1 , which leads to altered integrin-FAK signaling. Integrin α v (red color) is proposed to play a critical role in C1GALT1-mediated invasiveness. This pathway is coordinated with other C1GALT1-regulated pathways, such as receptor tyrosine kinases (RTKs) and mucins, to promote tumor growth and metastasis in pancreatic cancer. FN fibronectin, VN vitronectin.

Article Snippet: Antibodies against C1GALT1(F-31), FAK (C-20), and integrin α 5 (H-104) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Western Blot, Knockdown, Over Expression, Transfection, shRNA, Plasmid Preparation, Control, Expressing, Flow Cytometry, Negative Control, Functional Assay, Blocking Assay, Phospho-proteomics, Clinical Proteomics, Membrane, Protein Extraction, Extraction, Purification

Journal: Frontiers in Immunology

Article Title: 29-Color Flow Cytometry: Unraveling Human Liver NK Cell Repertoire Diversity

doi: 10.3389/fimmu.2019.02692

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: CD49e , REA686 , VioBright FITC , , 530/30 , 100 , No , Miltenyi biotec , Tissue residency/cell retention.

Techniques: Marker, Staining

Journal: Frontiers in Immunology

Article Title: 29-Color Flow Cytometry: Unraveling Human Liver NK Cell Repertoire Diversity

doi: 10.3389/fimmu.2019.02692

Figure Lengend Snippet: A 29-color NK-cell focused panel captures phenotypic diversity within liver-enriched NK cells. (A) Heatmap displays Z-score transformed median expression values for each of the parameters within 18 identified PhenoGraph clusters as described in . Color scale was determined for each column separately, based on the lowest and highest Z-score value of that parameter. (B) Histograms displaying expression levels of selected proteins in PhenoGraph clusters. Legend indicates whether a cluster is predominantly enriched in liver (#3), present in similar frequencies in liver and blood (shared, #18) or enriched in blood (#10, #12). (C) Histograms displaying expression levels of selected proteins from (A) in selected liver-enriched clusters (#4, #5, #15). (D) Identification of the two most proliferating PhenoGraph clusters (#3 and #14) according to their Ki-67 expression level based on analysis in (A) , displayed against CD49e.

Article Snippet: CD49e , REA686 , VioBright FITC , , 530/30 , 100 , No , Miltenyi biotec , Tissue residency/cell retention.

Techniques: Transformation Assay, Expressing