Journal: Scientific Reports

Article Title: Genistein enhances anti-PD-1 efficacy in melanoma by suppressing regulatory T cell differentiation and activity

doi: 10.1038/s41598-025-20941-7

Figure Lengend Snippet: Genistein reversed the immunosuppressive microenvironment in melanoma. ( A ) Quantification of tumor-infiltrating CD3⁺CD8⁺ cytotoxic T lymphocytes (Gating Strategy Fig. ). ( B ) Quantification of tumor-infiltrating CD4 + Foxp3 + Tregs. ( C ) Ratio of CD8 + cytotoxic T cells to Tregs. ( D ) Concentrations of IFN-γ, TNF-α, and IL-10 in tumor tissues measured by ELISA. ( n = 5). Data are presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The cell suspension was filtered through a 40 μm cell strainer, and splenocytes were isolated using the MagCellect Mouse CD4 + CD25 + Regulatory T Cell Isolation Kit (MAGM208, R&D systems, CA, USA) to obtain primary CD4 + T lymphocytes and CD4 + CD25 + Tregs.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Genistein enhances anti-PD-1 efficacy in melanoma by suppressing regulatory T cell differentiation and activity

doi: 10.1038/s41598-025-20941-7

Figure Lengend Snippet: Genistein dose-dependently impaired Treg function. ( A ) RT-qPCR analysis of Treg functional markers (Foxp3, CTLA-4, and LAG-3) after 24-h treatment with genistein at concentrations of 0–20 µM. ( B ) Western blot detection of Foxp3 protein expression following 24-h exposure to genistein (0–20 µM). ( C ) ELISA quantification of IL-10 in Treg culture supernatants after 24-hour genistein treatment (0–20 µM). ( D ) CD4 + CD25 − Th cells (2 × 10 5 /ml) were labeled with CFSE and subsequently cocultured with Tregs preincubated with genistein (10 µM) for 48 h. The Th cell proliferation was determined by flow cytometry. ( E ) ELISA quantification of IFN-γ and TNF-α in supernatants from Treg and Th cell co-cultures. ( n = 3). Data are showed as mean ± SD; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The cell suspension was filtered through a 40 μm cell strainer, and splenocytes were isolated using the MagCellect Mouse CD4 + CD25 + Regulatory T Cell Isolation Kit (MAGM208, R&D systems, CA, USA) to obtain primary CD4 + T lymphocytes and CD4 + CD25 + Tregs.

Techniques: Quantitative RT-PCR, Functional Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Labeling, Flow Cytometry

Journal: Scientific Reports

Article Title: Genistein enhances anti-PD-1 efficacy in melanoma by suppressing regulatory T cell differentiation and activity

doi: 10.1038/s41598-025-20941-7

Figure Lengend Snippet: Genistein suppressed Treg differentiation by targeting the PI3K-Akt pathway. ( A ) CD4 + CD25 − naïve T cells were differentiated into Tregs under anti-CD3/CD28 stimulation and 2 ng/ml TGF-β1 for 72 h, with or without 10 µM genistein. Flow cytometry was used to quantify CD4 + Foxp3 + cells. ( B ) qRT-PCR analysis of Treg signature genes ( Foxp3 , CTLA-4 , LAG-3 , IL-10 ) in cells treated as in ( A ). ( C ) Western blot detection of phosphorylated PI3K and AKT in the induced T cells, which were incubated with genistein (10 µM) or the combination of genistein (10 µM) and SC-79 (10 µM) under Treg-inducing conditions for 72 h. ( D ) Flow cytometric quantification of CD4 + Foxp3 + cells under the conditions in ( C ). ( E ) mRNA expression of Foxp3, CTLA-4, LAG-3 and IL-10 in induced T cells under the conditions in ( C ). Data are presented as mean ± SD ( n = 3); * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The cell suspension was filtered through a 40 μm cell strainer, and splenocytes were isolated using the MagCellect Mouse CD4 + CD25 + Regulatory T Cell Isolation Kit (MAGM208, R&D systems, CA, USA) to obtain primary CD4 + T lymphocytes and CD4 + CD25 + Tregs.

Techniques: Flow Cytometry, Quantitative RT-PCR, Western Blot, Incubation, Expressing

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Hypercholesterolemic Dysregulation of Calpain in Lymphatic Endothelial Cells Interferes With Regulatory T-Cell Stability and Trafficking

doi: 10.1161/atvbaha.122.317781

Figure Lengend Snippet: Figure 2. TGF (transforming growth factor)-β1–mediated stabilization of regulatory T cells (Tregs) on lymphatic endothelial cells is limited by lysophosphatidic acid (LPA)-induced disinhibition of conventional calpains. A, Treg stability on SVEC4-10 cells. Isolated splenic CD4+ cells were seeded on SVEC4-10 cell monolayer and cultured for 5 days in the presence or absence of LPA at 1 µmol/L (n=6 experiments). B, Expression levels of Treg-stabilizing factors in SVEC4-10 cells. Control and Capns1-knockdown cells were stimulated with LPA at 1 µmol/L for 6 hours. (Ctl: n=7 experiments, Ctl+LPA: n=8 experiments, siCapns1-1+LPA: n=8 experiments). C, Pharmacological assessment of Treg stabilization. Splenic CD4+ cells were cocultured with SVEC4-10 cells in the presence of a TGF-β type-I receptor antagonists SB431542 (3 µmol/L), SB525334 (3 µmol/L), LY-364947 (3 µmol/L), or IDO1 inhibitor INCB 024360 (1 µmol/L) for 5 days, concomitant with LPA at 1 µmol/L (Ctl: n=13 experiments, siCapns1-1: n=13 experiments, SB4: n=8 experiments, SB5: n=8 experiments, Ly36: n=5 experiments, INCB: n=5 experiments). D, Immunoblot analysis of intracellular TGF-β1 protein levels in SVEC4-10 cells (n=6 experiments) and human dermal lymphatic endothelial cells (HDLECs; n=4 experiments). Control and siRNA-transfected cells were stimulated with LPA at 1 µmol/L for 16 hours. E, Impact of LPA- induced calpain activation on phorbol 12-myristate 13-acetate (PMA)–induced TGF-β1 production in LECs. SVEC4-10 cells were treated with PMA at 200 nmol/L. LPA at 1 µmol/L was added as required (n=3 experiments). F, Pharmacological assessment of TGF-β1 synthesis pathways in SVEC4-10 cells. Cells were treated with an ERK inhibitor PD98059 at 50 µmol/L, JNK inhibitor SP600125 at 50 µmol/L, p38MAPK (mitogen-activated protein kinase) inhibitor SB203580 at 5 µmol/L, or a c-fos/activator protein 1 inhibitor T5224 at 50 µmol/L for 24 hours in the presence of LPA at 1 µmol/L (n=3 experiments). G, Phosphorylation of ERK and JNK in SVEC4-10 cells. Cells were cultured with LPA at 1 µmol/L for 24 hours (n=3 experiments). H, Stability of MEKK1 (mitogen-activated protein kinase kinase kinase 1) in SVEC4-10 cells. Cells were stimulated with LPA at 1 µmol/L for 24 hours (n=6 experiments). Data were expressed as median with 95% confidence limits. P values were determined by a Kruskal-Wallis test with post hoc Bonferroni test (A–H). ACTB indicates beta-actin; Ctl, control; ERK, extracellular signal-regulated kinase; FOXP3, forkhead box P3; INCB, INCB024360; JNK, c-Jun N-terminal kinase; MEKK, mitogen-activated protein kinase kinase kinase 1; PD, PD98059; SB, SB203580; SB4, SB431542; SB5, SB525334; Si, siRNA; siCapns1, siRNA against Capns1; SP, SP600125; T, T5224; pERK, phospho ERK; pJNK, phospho JNK; and Veh, vehicle.

Article Snippet: Then the cells were labeled by using a MagCellect Mouse CD4+ T-Cell Isolation Kit (R&D Systems, Inc) and were separated by magnetic fields using negative selection.

Techniques: Isolation, Cell Culture, Expressing, Control, Knockdown, Western Blot, Transfection, Activation Assay, Phospho-proteomics

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Hypercholesterolemic Dysregulation of Calpain in Lymphatic Endothelial Cells Interferes With Regulatory T-Cell Stability and Trafficking

doi: 10.1161/atvbaha.122.317781

Figure Lengend Snippet: Figure 5. Conventional calpains in lymphatic endothelial cells decelerate lymphatic trafficking through induction of VCAM1 (vascular cell adhesion molecule 1). A, Lymphocyte homing assay. Following 12 weeks of diet feeding, CFDA (5(6)-carboxyfluorescein diacetate)-labeled splenic CD4+ cells were injected into the footpad of mice. Sixteen hours after the injection, CD4+ T cells and CD4+FOXP3+ regulatory T cells (Tregs) in popliteal lymph nodes (LNs) were monitored (LNL [loxP-flanked Neo cassette]: n=9 animals, cTg: n=8 animals). B, Gene expression profiles in mesenteric LNs. The genes exhibiting high expression levels with a statistically significant difference are indicated in mean-average plot (left; n=3 animals). C, VCAM1 neutralization inhibits afferent lymphatic transportation of Tregs. VCAM1 antibody (2.5 µg/site) or nonimmune IgG (2.5 µg/site) was injected together with CD4+ cells (Cont IgG: n=5 animals, VCAM1 mAb: n=6 animals). D, Quantification of VCAM1 immunofluorescent intensity in lymphatic endothelial cells (LECs) in human coronary artery disease (CAD) and non-CAD cases (non-CAD case: n=77 cells from 5 cases, CAD case: n=63 cells from 3 cases). E, Immunohistochemical localization of VCAM1 in gp38+PROX1+ (prospero-related homeobox 1) lymphatic vessels in brachial LNs in normal laboratory diet (NLD)–fed or high cholesterol diet (HCD)–fed Ldlr–/– mice. Arrows represent LECs. Bar: 20 µm. VCAM1 fluorescence intensity in individual gp38+PROX1+ LECs was measured (LNL-CAST, n=34 cells from 3 animals; CAST cTg, n=32 cells from 3 animals; Capns1 flox, n=36 cells from 3 animals; Capns1 cKO, n=30 cells from 3 animals). Bar: 10 µm. Data in C and D were expressed as median with 95% confidence limits. Others were indicated as mean±SEM. P values were determined by Mann-Whitney test (C and D), Student t test (A [CD4+ cells], B, E [CAST]), or Welch test (A [Tregs] and E [Capns1]). A.U. indicates arbitrary unit; CAST, calpastatin; CFSE, 5(6)-carboxyfluorescein diacetate succinimidyl ester; FSC, forward scatter; mAb, monoclonal antibody; Neg Cont, negative control; and pLN, popliteal LNs.

Article Snippet: Then the cells were labeled by using a MagCellect Mouse CD4+ T-Cell Isolation Kit (R&D Systems, Inc) and were separated by magnetic fields using negative selection.

Techniques: Labeling, Injection, Gene Expression, Expressing, Neutralization, Immunohistochemical staining, Fluorescence, MANN-WHITNEY, Negative Control