Journal: Aging

Article Title: An integrated bioinformatic investigation of focal adhesion-related genes in glioma followed by preliminary validation of COL1A2 in tumorigenesis.

doi: 10.18632/aging.204834

Figure Lengend Snippet: Figure 9. COL1A2 inhibits the effect of activated Jurkat cells on the viability of GBM cells in vitro. (A) Immunofluorescence staining of the nucleus, COL1A2, and CD8 in eight cases of GBM tissues. (B, C) Jurkat cells were stimulated by PMA and ionomycin, or by anti- CD3 and anti-CD28 antibodies. After 24 hours of stimulation, the expression of CD8 and CD69 proteins were detected. (D, E) The viability of U87 (D) and U251 (E) cells were analyzed by measuring luciferase activity. ***P<0.001.

Article Snippet: Method 2: Jurkat cells were stimulated by adding 5μg/ml of anti-CD3 antibody (17617-1-AP, Proteintech, China), which was coated in a 6-well plate with the 5μg/ml of anti-CD28 antibody (65099-1-Ig, Proteintech).

Techniques: In Vitro, Immunofluorescence, Staining, Expressing, Luciferase, Activity Assay

Journal: Cell Reports Medicine

Article Title: OX40 agonism enhances PD-L1 checkpoint blockade by shifting the cytotoxic T cell differentiation spectrum

doi: 10.1016/j.xcrm.2023.100939

Figure Lengend Snippet:

Article Snippet: Anti-mouse CD28 (clone 37.51) (151Eu) , Fluidigm , 3151005B.

Techniques: Recombinant, Purification, Staining, Cell Isolation, Transgenic Assay, Software, Gene Expression

Journal: Journal of Biomedical Science

Article Title: Tumor-secreted IFI35 promotes proliferation and cytotoxic activity of CD8 + T cells through PI3K/AKT/mTOR signaling pathway in colorectal cancer

doi: 10.1186/s12929-023-00930-6

Figure Lengend Snippet: Tumor-secreted IFI35 promotes CD8 + T cells proliferation. A ELISA analysis in the supernatant of IFI35 overexpressed or knocked down CT26 and MC38 cells. B – G Effect of IFI35 protein on T cell migration, apoptosis and proliferation in vitro. T cell migration, apoptosis and proliferation in the presence of supernatant from murine colon cancer cells expressing shRNA against IFI35 (shIFI35) and scrambled sequence control (shRNA) for 72 h. In all experiments, mouse splenic CD8 + T cells were stimulated with plate-bound anti-CD3/CD28 mAbs. B , C Migration of CD8 + T cells by flow cytometric analysis. D , E Flow cytometric analysis of mouse splenic CD8 + T cells apoptosis. F , G Proliferation of CFSE-labeled mouse splenic CD8 + T cells by flow cytometric analysis. n = 3. Error bars represent the mean ± SEM. Two tailed t-tests, ns not significant. ***P < 0.001, ****P < 0.0001. H , I Effect of IFI35 on T cell proliferation in vivo. H Gating strategy for Ki67 + CD8 + T cells in CT26 tumors. I Quantification of Ki67 expression among CD8 + T cells in CT26 tumors. n = 4 for both groups. Two tailed t-tests. **P < 0.01

Article Snippet: For transduction of T cells, human PBMC was stimulated with CD3 and CD28 antibodies (Biogems, Westlake Village, CA, USA) for 24 h, and the lentiviral vector from above was added at an MOI of 5 IU/cell.

Techniques: Enzyme-linked Immunosorbent Assay, Migration, In Vitro, Expressing, shRNA, Sequencing, Control, Labeling, Two Tailed Test, In Vivo

Journal: Journal of Biomedical Science

Article Title: Tumor-secreted IFI35 promotes proliferation and cytotoxic activity of CD8 + T cells through PI3K/AKT/mTOR signaling pathway in colorectal cancer

doi: 10.1186/s12929-023-00930-6

Figure Lengend Snippet: Tumor-secreted IFI35 protein activated CD8 + T cells thought PI3K/AKT/mTOR pathway. A Representative Western blots of p-mTOR, mTOR, p-AKT, AKT, p-ERK, ERK, p-JNK, JNK, p-P38, P38, p-P65, P65, p-STAT3, STAT3, and GAPDH in CD8 + T cells. CD8 + T cells stimulated with anti-CD3 (1 µg/mL), anti-CD28 (1 µg/mL), and IL-2 (10 ng/mL) were treated with supernatant from IFI35 knockdown or overexpression CT26 and MC38 cells for 2 days. B, Representative western blots of p-mTOR, p-AKT, and GAPDH in CD8 + T cells. The CD8 + T cells stimulated with anti-CD3 (1 µg/mL), anti-CD28 (1 µg/mL), and IL-2 (10 ng/mL) were pretreated with or without chemical inhibitors Wortmannin (20 nM) at 37 °C for 2 h. C , D The CFSE-labeled mouse CD8 + T cells were pretreated with or without wortmannin (20 nM) for 2 h. The cells were stimulated with anti-CD3 (1 µg/mL), anti-CD28 (1 µg/mL), and IL-2 (10 ng/mL) in supernatant from IFI35 and control vector expressing CT26 for 72 h. Cell divisions were then analyzed by flow cytometry. n = 3. Error bars represent the mean ± SEM. Two tailed t-tests, ***P < 0.001, ****P < 0.0001. E – H Flow cytometric analysis of IFNγ and TNFα of CD8 + T cells. CD8 + T cells were pretreated with or without wortmannin (20 nM) for 2 h and stimulated with anti-CD3 (1 µg/mL), anti-CD28 (1 µg/mL), and IL-2 (10 ng/mL) in supernatant from IFI35 and control vector expressing CT26 for 72 h. n = 3. Error bars represent the mean ± SEM. Two tailed t-tests, ***P < 0.001, ****P < 0.0001

Article Snippet: For transduction of T cells, human PBMC was stimulated with CD3 and CD28 antibodies (Biogems, Westlake Village, CA, USA) for 24 h, and the lentiviral vector from above was added at an MOI of 5 IU/cell.

Techniques: Western Blot, Knockdown, Over Expression, Labeling, Control, Plasmid Preparation, Expressing, Flow Cytometry, Two Tailed Test