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. 2014 Mar 25;124(5):2076–2086. doi: 10.1172/JCI71194

Figure 2. S1P1 activation in MEFs.

Figure 2

(A) Validation of modified S1P1 signaling pathway in MEFs. S1P1 GFP signaling MEFs were cultured for 16 hours in medium containing 10% charcoal-stripped FBS and received either S1P (10–7 M) or vehicle (4 mg/ml BSA in PBS). Nuclei were stained with Hoechst, and MEF cultures were imaged under an inverted laser-scanning confocal microscope. The experiment was repeated twice in duplicate, and a representative result is shown. (B) Treatment of MEFs with RP-001. S1P1 GFP signaling MEFs were cultured for 16 hours in medium containing 10% charcoal-stripped FBS and received either S1P or RP-001. After 24 hours, nuclei were stained with Hoechst, and MEF cultures were imaged under an inverted laser-scanning confocal microscope. The experiment was performed in duplicate, and a representative result is shown. (C) Flow cytometry analysis of S1P1 GFP signaling MEFs. S1P1 GFP signaling MEFs were cultured for 16 hours in medium containing 10% charcoal-stripped FBS and various concentrations of S1P were added. After 24 hours, the number of GFP+ cells was determined by flow cytometry. The experiment was repeated twice. Data represent mean ± SEM (n = 3). (D) Endogenous S1P1 signaling pathway in MEFs. S1P1 GFP signaling MEFs were cultured for 16 hours in medium containing 0.1% FBS and received 1 μM of S1P1 receptor ligands (RP-001, S1P, or SEW2871) or vehicle (4 mg/ml BSA in PBS). After 10 minutes, the cell lysate was harvested and then Akt and phospho-Akt were identified by Western blotting (see complete unedited blot in the supplemental material). The experiment was performed in triplicate, and a representative result is shown. Scale bars: 100 μm.