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. 2014 Apr 7;211(4):685–700. doi: 10.1084/jem.20131343

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© 2014 Willinger et al.

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Figure 4. — Dynamin 2 is necessary for ligand-induced S1PR1 internalization at low S1P concentrations. (A) S1PR1 surface expression on mature CD4 SP thymocytes and naive (CD44^lo) CD4 T cells from Dnm2 HET and KO cells (n > 10) was measured by flow cytometry. To define background S1PR1 staining, control mice were injected i.p. with FTY720 16 h before flow cytometry. (B) S1PR1 surface expression on splenic naive CD4 T cells from Dnm2 HET/B6 and Dnm2 KO/B6 mixed bone marrow chimeras (n = 6) generated as in Fig. 1 G. Green histograms represent background S1PR1 staining (thymocytes from control chimeras injected with FTY720). (C) Dnm2 HET and KO mice were injected i.p. with PBS or FTY720 (2 mg/kg body weight). S1PR1 surface expression on mature CD4 SP thymocytes (n = 6) was measured by flow cytometry 16 h later. Green histograms represent S1PR1 staining on DP thymocytes as negative control. (D and E) S1PR1 surface expression on mature CD4 SP thymocytes from Dnm2 HET and KO mice treated with PBS or the indicated concentrations (per body weight) of FTY720 16 h before analysis (n = 3). (F and G) Dnm2 HET and KO mice received 30 mg/liter DOP in the drinking water or water alone. 3 d later, S1PR1 surface expression on mature CD4 SP thymocytes (n = 7) was measured by flow cytometry. Green histograms represent background S1PR1 staining (thymocytes from mice injected with FTY720). (H) S1PR1 surface expression on mature CD4 SP thymocytes from Dnm2 HET and KO mice treated in vitro with the indicated concentrations of S1P (n = 3–7). All error bars represent SEM. *, P < 0.05; **, P < 0.01 by unpaired Student’s t test. Results are representative of or combined from two (B, F, and G), three (C–E and H), or four (A) experiments.