Journal: PLOS One

Article Title: Identification of Galectin-9 (Gal-9) as a B7-H4 binding partner and characterization of their glycosylation-dependent interaction that modulates T cell signaling within a multi-ligand/receptor network

doi: 10.1371/journal.pone.0355964

Figure Lengend Snippet: (A) SPR analysis of Gal-9 binding kinetics to PD-1, TIM-3, CD28, and B7-H4. Human PD-1, TIM-3, and CD28 were fused to the same mouse IgG2a Fc tags. 1.2 µg/mL Gal-9 was injected over a Protein A/G-coated Biacore chip to assess binding kinetics to each immobilized Fc-fusion protein. (B) Flow cytometry analysis of the binding between the four proteins (shown in Panel A) and 293T cells transiently overexpressing either WT Gal-9 or the Gal-9 R65A mutant. ( C ) Cell-surface binding of CD28 to Gal-9 analyzed by Co-IP-Western blotting. Cell-surface expression of CD28 on Jurkat T cells was examined by flow cytometry (left panel); CD28 expression was detected using an anti-CD28 antibody (red), with a cell-only control included (black). For the Co-IP-Western blotting analysis (right panel); 400 µL of whole cell lysates (WCL) from Jurkat T cells were incubated with or without 4 µg/mL Gal-9 and 200 mM lactose, followed by IP using an anti-CD28 antibody. Bound proteins were eluted and analyzed by Western blotting using a different anti-CD28 antibody from that used for IP, together with an anti-Gal-9 antibody, as indicated. ( D ) Cell-surface binding of B7-H4 to Gal-9 was analyzed by Co-IP-Western blotting. Cell-surface expression of B7-H4 on 293T cells transiently transfected with B7-H4 (293T-B7-H4) was examined by flow cytometry (left panel); B7-H4 expression was detected with an anti-B7-H4 antibody (blue), with a cell-only control included (black). For the Co-IP-Western blotting analysis (right panel), 400 µL of WCL from 293T-B7-H4 cells were incubated with or without 4 µg/mL Gal-9 and 200 mM lactose, followed by IP using an anti-B7-H4 antibody. Bound proteins were eluted and analyzed by Western blotting using a different anti-B7-H4 antibody from that used for IP, and an anti-Gal-9 antibody, as indicated. Anti-GAPDH antibody was used as an internal loading control for Western blotting of WCL samples in (C) and (D). (E) Gal-9-mediated stimulation of pCD28 signaling activity in Jurkat T cells. Jurkat T cells were treated with or without plate-bound B7.1, and Gal-9’s dose-dependent effects on pCD28 were analyzed by western blotting. Fold change (FC) in pCD28 band intensity relative to the CD28 loading control was quantified using ImageJ and GraphPad Prism.

Article Snippet: Antibodies and reagents used for western blotting were primarily obtained from ABclonal unless otherwise indicated: Phospho-CD28 (pCD28; Y191) Rabbit mAb (Cell Signaling Technology, 16399), CD28 Rabbit mAb (A20346), Phospho-Akt (pAKT; S473) Rabbit mAb (AP1208), Pan-Akt Rabbit mAb (A18675), HRP-conjugated β-Actin Rabbit mAb (AC028), Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology, 5872), and Lactose (LABLEAD BIOTECH, 5989-81-1).

Techniques: Binding Assay, Injection, Flow Cytometry, Mutagenesis, Co-Immunoprecipitation Assay, Western Blot, Expressing, Control, Incubation, Transfection, Activity Assay

Journal: PLOS One

Article Title: Identification of Galectin-9 (Gal-9) as a B7-H4 binding partner and characterization of their glycosylation-dependent interaction that modulates T cell signaling within a multi-ligand/receptor network

doi: 10.1371/journal.pone.0355964

Figure Lengend Snippet: (A-C) B7-H4 modulates pCD28 and pAKT signaling in T cells in a glycosylation-dependent manner. Jurkat (A), MOLT-4 (B), and primary T cells (C) were treated with 8 µg/mL Gal-9 in the presence or absence of 25 µg/mL hB7-H4-Fc, hB7-H4 IgV-Fc, or de-glycosylated B7-H4 MGAT1- . Western blotting was performed to detect pCD28 and pAKT signaling activity (top panels). Fold changes (FC) in pCD28 and pAKT band intensity relative to the corresponding CD28 and AKT loading controls were quantified using Image J and GraphPad Prism (middle and bottom panels, respectively). Gal-9-mediated stimulation of pCD28 and pAKT signaling, and its regulation by hB7-H4-Fc, were independently replicated in Jurkat (Panel A and ), MOLT-4 T cells (Panel B and ), and primary T cells (Panel C and ) in two or three experiments. (D) Gal-9 stimulates NFAT signaling. Jurkat-NFAT-Luc reporter cells showed dose-dependent luciferase induction by Gal-9, but not hB7-H4-Fc. (E) B7-H4 attenuated Gal-9-induced CD28 signaling. Jurkat-NFAT-Luc reporter cells were treated with 3.3 µg/mL Gal-9, 25 µg/mL hB7-H4-Fc, or both. (F) Flow cytometry analysis of B7-H4 expression on 293F cells. B7-H4 expression on 293F cells (293F-B7-H4) was confirmed, the 293F-B7-H4 cells were used in panel G. (G) Cell-surface B7-H4 attenuated Gal-9-induced CD28 signaling. Jurkat-NFAT-Luc reporter cells were co-cultured with 293F or 293F-B7-H4 cells in the presence or absence of 3.3 µg/mL Gal-9. One-way ANOVA was used to statistically analyze the mean of NFAT-mediated luminescence signals among groups in Panels E and G. All data are presented as mean ± s.e.m.

Article Snippet: Antibodies and reagents used for western blotting were primarily obtained from ABclonal unless otherwise indicated: Phospho-CD28 (pCD28; Y191) Rabbit mAb (Cell Signaling Technology, 16399), CD28 Rabbit mAb (A20346), Phospho-Akt (pAKT; S473) Rabbit mAb (AP1208), Pan-Akt Rabbit mAb (A18675), HRP-conjugated β-Actin Rabbit mAb (AC028), Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology, 5872), and Lactose (LABLEAD BIOTECH, 5989-81-1).

Techniques: Glycoproteomics, Western Blot, Activity Assay, Luciferase, Flow Cytometry, Expressing, Cell Culture

Journal: Molecular Therapy Oncology

Article Title: Innovative photodynamic therapy using rose bengal for the treatment of human melanoma

doi: 10.1016/j.omton.2026.201241

Figure Lengend Snippet: Evaluation of immunologic response with PDT-RB (A–D) Proliferation of activated PBMCs (anti-CD3 – anti-CD28 cocktail) in co-culture with conditioned media derived from HBL (A and C) and LND (B and D) cell lines, respectively, after 72 h and 120 h of culture. Results are presented as mean ± SEM of 4 independent experiments, one-way ANOVA statistical test was performed with ∗ p < 0.05, ∗∗ p < 0.001 being considered statistically significant. NT, non-treated cells; Illu, illuminated alone without Rose Bengal; RB, Rose Bengal; PDT-RB, cells incubated with RB and illuminated.

Article Snippet: PBMCs were either stimulated or not stimulated with anti-CD3 (0.25 μg/mL; Miltenyi, Bergisch Glad bach, Germany) and anti-CD28 (0.25 μg/mL; Clinisciences, Montrouge, France).

Techniques: Co-Culture Assay, Derivative Assay, Incubation