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. 2013 Apr 15;288(21):14839–14851. doi: 10.1074/jbc.M112.426726

FIGURE 6.

FIGURE 6.

Analysis of S1P2,3R subtype signaling in NHLF using siRNA knockdown and impedance assays. A, relative mRNA expression changes of S1P2R and S1P3R in NHLF 48 h after transfection with negative control (neg. co) siRNA, S1P2R siRNA a, S1P2R siRNA b, S1P3R siRNA a, or S1P3R siRNA b compared with nontransfected NHLF (untreated). Expression of NHLF transfected with negative control siRNA was set to 100%. The data represent the means ± S.E. of two independent experiments. B and C, NHLF were transfected with negative control siRNA (neg.; B and C), two S1P3R siRNAs (S1P3 a and S1P3 b; B), or two S1P2R siRNAs (S1P2 a and S1P2 b; C). After 48 h, NHLF transfected with negative control siRNAs were preincubated with or without S1P2R (C) or S1P3R (B) antagonists for 1 h. Then all samples were stimulated with vehicle, ponesimod (5000 nm; B and C), or FTY720-P (5 nm, B; 500 nm, C). Signaling was monitored over 2–5 h to confirm S1P2R and S1P3R subtype signaling deciphered with pharmacological S1PR antagonists. The data show representative experiments (n = 3).