Journal: Advanced Science
Article Title: Steroids Enable Mesenchymal Stromal Cells to Promote CD8 + T Cell Proliferation Via VEGF‐C
doi: 10.1002/advs.202003712
Figure Lengend Snippet: VEGF‐C augments CD8 + T cell proliferation via activating the PI3K/AKT signaling pathway. A) GO analysis showing the enriched pathways in CD8 + T cells co‐cultured with MSCs plus anti‐CD3 and anti‐CD28, in presence or absence of Dex. B) Heatmap showing genes associated with cell cycle in activated CD8 + T cells, with or without the addition of Dex or MSCs. C) Immunoblotting analysis of Cyclin D1, Cdk2, Cdk4, and Cdk6 in lysates of CD8 + T cells stimulated with anti‐CD3 and anti‐CD28, with or without addition of VEGF‐C for 24 h. D) Immunoblotting analysis of p85/p55, Akt, and their phosphorylation in lysates of CD8 + T cells stimulated with anti‐CD3 and anti‐CD28, with or without the addition of VEGF‐C. E) Inhibition of PI3K/AKT signaling abolished the promotion of VEGF‐C on CD8 + T cell proliferation. CD8 + T cells were isolated and stimulated with anti‐CD3 and anti‐CD28, with or without VEGF‐C in the presence or absence of LY294002 (20 µ m ) or Afuresertib (1 µ m ) for 48 h. Cell proliferation was assessed by 3 H‐thymidine incorporation. F) Immunoblotting analysis of p85/p55, Akt and their phosphorylation in lysates of CD8 + T cells derived from VEGFR3 fl/fl and VEGFR3 fl/fl CD8 Cre mice. Data are presented as mean ± SEM. ** p < 0.01.
Article Snippet: Protein samples were separated by SDS‐PAGE and transferred onto PVDF membranes, which was then blocked with 5% fat‐free milk for 1 h. Blots were incubated with primary antibodies specific to p‐p85/p‐p55, p85, p‐Akt, Akt, Cyclin D1, Cdk2, Cdk4, and Cdk6 (Cell Signaling Technology, USA) followed by secondary antibodies conjugated with horseradish peroxidase, and the staining was detected with the ECL system (Millipore).
Techniques: Cell Culture, Western Blot, Phospho-proteomics, Inhibition, Isolation, Derivative Assay
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