Journal: The Journal of Clinical Investigation

Article Title: S1PR4 ablation reduces tumor growth and improves chemotherapy via CD8 + T cell expansion

doi: 10.1172/JCI136928

Figure Lengend Snippet: (A) Experimental outline of the AOM/DSS model applied to WT and S1PR4-KO mice. (B) Weight of AOM/DSS-treated WT and S1PR4-KO mice as a percentage of weight at the initiation of treatment (n = 9). (C and D) Relative amounts of CD45+ leukocytes (C) and immune cell populations (D) in the LP of WT and S1PR4-KO mice at day 0 (n = 6), day 8 (n = 8), day 15 (n = 4), and day 84 (n = 9) analyzed by flow cytometry. (E) Representative pictures of WT and S1PR4-KO AOM/DSS-treated colon tissue at day 8 stained with H&E. Scale bars: 1 mm; scale bars of magnified areas:100 μm. (F) Colon weight-to-length ratio determined for WT and KO AOM/DSS-treated mice at days 0 (WT: n = 7, KO: n = 8), 8 (n = 4), 15 (n = 5), and 84 (n = 10). Means ± SEM; 2-tailed Student’s t test; *P < 0.05.

Article Snippet: Mice were i.p. injected with 10 mg/kg AOM (MilliporeSigma) at day 0 and day 21 before receiving 0.5% DSS (MP Biomedicals) for the following 5 days in the drinking water.

Techniques: Flow Cytometry, Staining

Journal: The Journal of Clinical Investigation

Article Title: S1PR4 ablation reduces tumor growth and improves chemotherapy via CD8 + T cell expansion

doi: 10.1172/JCI136928

Figure Lengend Snippet: (A) Representative images of WT and S1PR4-KO AOM/DSS-treated colons at day 84 stained with H&E. Scale bars: 1 mm. (B) Number of tumors per mouse for WT and S1PR4-KO mice (n = 9) at day 84 after treatment with AOM/DSS. (C) Representative tSNE plots showing the composition of the epithelial layer from WT and S1PR4-KO colons at day 84. (D and E) Relative amounts of CD8+ IEL (D) and CD8+ Trm IELs (E) within the epithelial layer of WT and S1PR4-KO mice at day 0 (n = 6), day 8 (n = 8), day 15 (n = 4), and day 84 (n = 9) analyzed by flow cytometry. Means ± SEM; 2-tailed Student’s t test; *P < 0.05, **P < 0.01.

Article Snippet: Mice were i.p. injected with 10 mg/kg AOM (MilliporeSigma) at day 0 and day 21 before receiving 0.5% DSS (MP Biomedicals) for the following 5 days in the drinking water.

Techniques: Staining, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: S1PR4 ablation reduces tumor growth and improves chemotherapy via CD8 + T cell expansion

doi: 10.1172/JCI136928

Figure Lengend Snippet: (A) Venn diagram and the gene list show shared and divergent up- or downregulated genes in PyMT tumor-derived CD8+ T cells and total AOM/DSS-treated colons (day 84) comparing WT and S1PR4-KO mice. Genes selected for in vitro validation are highlighted in green. (B and C) Absolute number of WT and S1PR4-KO CD8+ T cells either untreated (w/o) or treated with (B) 0.5 μM PI3K inhibitor (Ly294002) or (C) 5 μM LTA4H inhibitor (SC 57461A) at day 2. One representative experiment with 5 independent biological replicates is shown, which was repeated 3 times with similar outcomes. (D–I) PyMT tumor spheroids were cocultured with WT and S1PR4-KO CD8+ T cells. One representative experiment with 5 independent biological replicates (each containing means of 6 technical replicates) is shown. (D–F) PyMT spheroid size upon coculture with untreated (black), Ly294002-treated (green), or SC 57461A–treated (red) CD8+ T cells over 6 days (D and E) and at day 6 (F) after initial activation with representative photographs (G–I). Scale bars: 200 μm. (J) Intracellular staining of p-AKT in NTC or PIK3AP1 siRNA-treated WT and S1PR4-KO CD8+ T cells 30 minutes after activation (n = 4). p-AKT expression is shown as MFI. (K) LTB4 concentration in supernatants of WT and S1PR4-KO CD8+ T cells 1 day after activation determined by ELISA (n = 5). (L and M) Absolute number of S1PR4 agonist (Cym 50308) or antagonist (Cym 50358) pretreated CD8+ T cells either untreated (w/o) or treated with 20 μM PGP 6 days (L) or 8 days (M) after activation (n = 5). Means ± SEM; 1-way ANOVA (B–F and J) or 2-way ANOVA (L and M), each with Holm-Šidák correction; *P < 0.05, **P < 0.01.

Article Snippet: Mice were i.p. injected with 10 mg/kg AOM (MilliporeSigma) at day 0 and day 21 before receiving 0.5% DSS (MP Biomedicals) for the following 5 days in the drinking water.

Techniques: Derivative Assay, In Vitro, Activation Assay, Staining, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay