Journal: PLoS ONE

Article Title: Novel PDE4 Inhibitors Derived from Chinese Medicine Forsythia

doi: 10.1371/journal.pone.0115937

Figure Lengend Snippet: A. 5×10 5 RAW264.7 cells were seeded in 96 wells for 18 h. Cells were primed with compounds at different concentration for 3 h before treated with LPS (1 ng/ml) for additional 8 h. TNF cytokine releases were monitored by ELISA. B. PBMC (0.2 ml at 1×10 5 /ml) were primed with compounds at different concentration for 3 h before treated with LPS (1 ng/ml) for additional 8 h. TNF cytokine releases were monitored by ELISA. % of TNF secretion were calculated and graphed. C. Summary of compound IC 50 . The data represent n = 3–6 experiments.

Article Snippet: IL1β, TNFα, IL6 mouse ELISA kit, human TNFα were from R&D systems.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Intratumoral CXCL13+ CD160+ CD8+ T cells promote the formation of tertiary lymphoid structures to enhance the efficacy of immunotherapy in advanced gastric cancer

doi: 10.1136/jitc-2024-009603

Figure Lengend Snippet: Targeting PDXK could promote the formation of TLSs and enhance the efficacy of immunotherapy in gastric cancer. ( A ) Heatmap displaying the metabolic feature for T-cell clusters using scMetabolism package. ( B ) Dot plot showing the expression of the genes encoding rate-limiting enzymes of vitamin B 6 metabolism in different types of T cells. Dot size encodes the percentage of cells expressing the gene, color encodes the average per cell gene expression level. ( C ) Quantification of CXCL13 in PDTFs in the presence of different enzyme inhibitors measured by ELISA. ( D ) Representative H&E staining and PDXK immunohistochemistry of gastric cancer tissues with different responses following immunotherapy. Scale bar, 500 µm. ( E ) The schematic diagram of the animal experiments. ( F–G ) Images of tumors and tumor volume curves of 615 mice treated with various agents (n=6, each group). ( H ) Paraffin sections of mouse subcutaneous graft tumor tissue stained with H&E and IHC detection for CD8, CD20 and CXCL13. Scale bar, 100 µm. ( I ) The number (left panel) and area (right panel) of TLS per tumor area were compared between groups (n=6, each group). Data are presented as the mean±SD. ns, not significant. *p<0.05, ***p<0.001, two-tailed Student’s t-test. AOX1, aldehyde oxidase 1; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CR, complete response; MFC, mouse forestomach carcinoma; PBS, phosphate-buffered saline; PDXK, pyridoxal kinase; PDXP, pyridoxal phosphatase; PDTFs, patient-derived tumor fragments; PHOSPHO2, phosphatase orphan 2; PNPO, pyridoxamine 5'-phosphate oxidase; PR, partial response; PSAT1, phosphoserine aminotransferase 1; s.c, subcutaneous injections; SD, stable disease; TLS, tertiary lymphoid structures.

Article Snippet: The indicated cytokines and chemokines within the supernatants were detected using human IL-2 ELISA Kit (Solarbio, SEKH-0008), human IFN-γ ELISA Kit (Solarbio, SEKH-0046), human CXCL13 ELISA Kit (Solarbio, SEKH-0072) and human TNF-α ELISA Kit (Solarbio, SEKH-0047) according to the manufacturers’ instructions.

Techniques: Expressing, Gene Expression, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Two Tailed Test, Saline, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Intratumoral CXCL13+ CD160+ CD8+ T cells promote the formation of tertiary lymphoid structures to enhance the efficacy of immunotherapy in advanced gastric cancer

doi: 10.1136/jitc-2024-009603

Figure Lengend Snippet: Vitamin B 6 could promote the expression and secretion of CXCL13 in CD160 + CD8 + T cells. ( A ) Quantification of cytokine/chemokine including CXCL13, IL-2, TNF-α and IFN-γ, in PDTFs in the presence of different drugs measured by ELISA. ( B ) The schematic diagram of orthotopic transplanted tumor model with diets containing various amounts of vitamin B 6 (n=6, each group). ( C ) Mouse orthotopic stomach xenograft tumor tissues stained with H&E and IHC detection for CD20 and CXCL13. Scale bar, 500 µm. ( D ) The schematic diagram of orthotopic transplanted tumor model fed with different drugs or diets (n=6, each group). ( E ) The representative images of mouse bioluminescence imaging at week 3 (left panel) and the corresponding quantification analysis (right panel). ( F ) Representative micrographs of xenografts stained with H&E and IHC detection for CD20 and CXCL13. Scale bar, 500 µm. ( G–H ) Density of TLSs (left panel) and ratio of tumor area occupied by TLSs (right panel). ( I ) Gating strategy for CD160 + CD8 + T cells. ( J ) Quantification of cytokine/chemokine including CXCL13, IL-2, TNF-α and IFN-γ, in supernatants from CD160 + CD8 + T-cell cultures in the presence or absence of PL measured by ELISA. ( K ) Flow cytometric analysis and corresponding quantification of CXCL13 + CD160 + CD8 + T cells with or without PL treatment. Data are presented as the mean±SD. ns, not significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-tailed Student’s t-test. ICIs, immune checkpoint inhibitors; IFN, interferon; IHC, immunohistochemistry; IL, interleukin; MFC, mouse forestomach carcinoma; PBS, phosphate-buffered saline; PL, pyridoxol; PD-1, programmed cell death protein 1; s.c, subcutaneous injections; TLS, tertiary lymphoid structures; TNF, tumor necrosis factor.

Article Snippet: The indicated cytokines and chemokines within the supernatants were detected using human IL-2 ELISA Kit (Solarbio, SEKH-0008), human IFN-γ ELISA Kit (Solarbio, SEKH-0046), human CXCL13 ELISA Kit (Solarbio, SEKH-0072) and human TNF-α ELISA Kit (Solarbio, SEKH-0047) according to the manufacturers’ instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Imaging, Two Tailed Test, Immunohistochemistry, Saline

Journal: Cancer Research

Article Title: Human 3D Ovarian Cancer Models Reveal Malignant Cell–Intrinsic and –Extrinsic Factors That Influence CAR T-cell Activity

doi: 10.1158/0008-5472.CAN-23-3007

Figure Lengend Snippet: Impaired death receptor signaling in malignant cells caused resistance to CAR T-cell cytotoxicity. A, Hierarchical cluster analysis of transcriptomes for OvCAR3 and G164 cells in monolayer and spheroids. B, Heatmap illustrating normalized gene expression of differentially expressed genes relating to death receptor signaling in OvCAR3 and G164 cells (adjusted P value < 0.05). C, cIAP1/2 expression on human HGSOC omental metastasis ( n = 16) and adjacent omentum ( n = 10). Statistics performed using two-way ANOVA. D and E, Representative images (left) and quantification (right) of Incucyte killing assay in which monolayers of G164 ( D ) and G33 ( E ) cells were treated with birinapant and CAR T cells. F, Western blot showing the expression of cIAP2 in OvCAR3 and G164 cells treated with birinapant. Representative images of three repeats. G, ELISA data showing TNFα concentration after coculturing CAR T cells with monolayers of OvCAR3 and G164 cells for 2 days at 1:5 T:E ratios. Data plotted as mean ± SD for three CAR T-cell donors. Statistics performed using two-way ANOVA. H, Representative images (left) and quantification (right) of Incucyte killing assay in which OvCAR3 monolayer was treated with anti-TNFα antibody and CAR T cells. D–F, Data shown for one CAR T-cell donor at 1:5 T:E ratio. Images shown are 3 days after treatment. Red, dead cells. Scale bars, 400 μm.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) was performed using human IFNα Quantikine kit (R&D Systems, Cat. DIF50C), human TNFα QuantiGlo kit (R&D Systems, Cat. QTA00C), human CCL2/MCP1 Quantikine kit (R&D Systems, Cat. DCP00), and human TGFβ1 Quantikine kit (R&D Systems, Cat. DB100C) according to manufacturer’s instructions.

Techniques: Gene Expression, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Cancer Research

Article Title: Human 3D Ovarian Cancer Models Reveal Malignant Cell–Intrinsic and –Extrinsic Factors That Influence CAR T-cell Activity

doi: 10.1158/0008-5472.CAN-23-3007

Figure Lengend Snippet: Vascularized microfluidic chip to investigate CAR T-cell migration and cytotoxicity. A, Design of the tri-channel microfluidic device with a 2 mm well in the central channel. B, Schematic diagram showing the development of ovarian cancer-on-a-chip model. Scale bar, 600 μm. C, Immunofluorescence image showing microvasculature in fibrin and OvCAR3 collagen gels ( n = 3). Red, HUVEC. Scale bar, 100 μm. D, Real-time images showing the luminal flow of CAR T cells (arrows) through the vasculature formed within the microfluidic device ( n = 2). Green, CAR T cells. Scale bar, 20 μm. E, Immunofluorescence image showing an ovarian cancer-on-a-chip 3 days after CAR T-cell treatment. CAR T cells (arrows) migrated into the OvCAR3 gel in the middle of the device. Dotted line marks the edge of the central well. Green, CAR T cells; red, HUVEC. Scale bar, 100 μm. F and G, CD3 ( F ) and caspase‐3 (Casp3; G ) staining and quantification of OvCAR3 gels isolated from microfluidic device after CAR T-cell and anti-TNFα treatment. H, MSD data showing TNFα, IFNγ, and IL2 concentrations on media from microfluidic devices after CAR T-cell and anti-TNFα treatment. F–H, Data plotted as mean ± SD of two/three gels per two replicates. Two different CAR T-cell donors were used in this experiment. Scale bar, 50 μm. Statistics performed using two-way ANOVA.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) was performed using human IFNα Quantikine kit (R&D Systems, Cat. DIF50C), human TNFα QuantiGlo kit (R&D Systems, Cat. QTA00C), human CCL2/MCP1 Quantikine kit (R&D Systems, Cat. DCP00), and human TGFβ1 Quantikine kit (R&D Systems, Cat. DB100C) according to manufacturer’s instructions.

Techniques: Migration, Immunofluorescence, Staining, Isolation

Journal: The Indonesian Biomedical Journal

Article Title: TNFR, TRAF2, NF-κB mRNA Levels of Glioblastoma Multiforme Cells Treated by Conditioned Medium of Umbilical Cord-derived Mesenchymal Stem Cells

doi: 10.18585/inabj.v11i2.722

Figure Lengend Snippet: Figure 5. TNFR2 level protein. Protein sample in the amount of 100 μL was used to measure TNFR2 level using ELISA with sandwich ELISA method. Protein TNFR2 level concentration was measured with absorbance 450 nm. TNFR2 concentration in CM treated T98G cells was 11.57 pg/mg protein while concentration in control was 7.72 pg/mg protein **(p=0.001). All data displayed in mean±SE (n=6).

Article Snippet: 2019; 11(2): 217-24DOI: 10.18585/inabj.v11i2.722 Enzyme Linked Imunoabsorbance Assay (ELISA) of TNF-α and TNFR2 TNF-α level from conditioned medium was measured using sandwich ELISA method with human ELISA TNF-α assay kit (Catalogue #E-EL-H0109, Elabscience, Texas, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Sandwich ELISA, Concentration Assay, Control

Journal: NPJ Regenerative Medicine

Article Title: Alkaline shear-thinning micro-nanocomposite hydrogels initiate endogenous TGFβ signaling for in situ bone regeneration

doi: 10.1038/s41536-023-00333-z

Figure Lengend Snippet: After 1-week treatment, decalcified bone tissue sections at rat calvarial defects in the coronal plane were prepared. a Representative H&E staining indicating the histological structures of repaired bone tissues by 10-MmN. Scale bar: 500 μm. b , c Immunohistochemical investigations showing ( b ) TGFβ-receptor 2 + (TGFβR2 + ) cells and ( c ) Ki67 + proliferating cells. Scale bar: 500 μm for Low mag and 100 μm for High mag. d Representative immunofluorescence images showing the positive staining for MSC surface markers of CD90 (green) and CD146 (red). Scale bar: 100 μm for Low mag (left) and 50 μm for High mag (right). Blue DAPI. MS (undegraded) gelatin microspheres. B bone tissue.

Article Snippet: For immunofluorescence, the primary antibodies include CD90 (ab181469, Abcam) and CD146 (ab75769, Abcam, Supplementary Table ).

Techniques: Staining, Immunohistochemical staining, Immunofluorescence