Figure 1.
Figure 1. Reciprocal control of S1P chemotaxis by counteracting the receptors S1PR1 and S1PR2. (A) Scheme of function and signal transduction of S1PR1 and S1PR2. (B) In vitro chemotactic response of BM-MDM isolated from wild-type and S1PR2-deficient mice. Before the chemotaxis assay, some cells were treated with 100 nM pertussis toxin (PTX). Error bars represent SD (n = 6, from three independent experiments). (C) In vitro S1P-directed chemotaxis of RAW264.7 cells dynamically visualized using EZ-Taxiscan. Cells were loaded in one side of the chamber and the other side was filled with medium containing indicated concentration of S1P (Videos 1–3). Cells migrate into the terrace between the loading chambers. The height from floor to ceiling of the terrace is 8 µm. Bar, 100 µm. (D) Tracking courses from the start line of representative cells in low, medium, and high S1P conditions. Each curve shows the data from one experiment and represents the averaged tracking distance of multiple cells over time. The EZ-Taxiscan experiments were independently performed six times and the data were largely consistent, although the extent of the toward-and-away motions of cells in 10−7 M S1P was variable depending on the experiment. Obvious away motion could clearly be observed in five of the six experiments (62 in 83 total cells), and the cells simply stopped in the middle of the chamber without clear backward migration in one of the six experiments (11 in 83 cells). (E) In vitro S1P-directed chemotaxis of RAW264.7 cells treated with siRNAs. Cells pretreated with control RNA duplex, siRNA against S1PR1, or siRNA against S1PR2 (Videos 4–6) were loaded into the EZ-Taxiscan chamber filled with a high concentration of S1P (10−9 M) in the other side. (F) RT-PCR detection of S1PR1 and S1PR2 in siRNA-pretreated RAW264.7 cells. Cells were pretreated with control RNA duplex (control siRNA), siRNA against S1PR1 (S1PR1 siRNA), or siRNA against S1PR2 (S1PR2 siRNA). (G) Migration distance data from microscopic analysis of control (red), S1PR1 knockdown (green), and S1PR2 knockdown (blue) cells. The experiments were independently performed three times and the data were largely consistent. Each dot represents the mean value of six independent cells and error bars represent SD.

Reciprocal control of S1P chemotaxis by counteracting the receptors S1PR1 and S1PR2. (A) Scheme of function and signal transduction of S1PR1 and S1PR2. (B) In vitro chemotactic response of BM-MDM isolated from wild-type and S1PR2-deficient mice. Before the chemotaxis assay, some cells were treated with 100 nM pertussis toxin (PTX). Error bars represent SD (n = 6, from three independent experiments). (C) In vitro S1P-directed chemotaxis of RAW264.7 cells dynamically visualized using EZ-Taxiscan. Cells were loaded in one side of the chamber and the other side was filled with medium containing indicated concentration of S1P (Videos 1–3). Cells migrate into the terrace between the loading chambers. The height from floor to ceiling of the terrace is 8 µm. Bar, 100 µm. (D) Tracking courses from the start line of representative cells in low, medium, and high S1P conditions. Each curve shows the data from one experiment and represents the averaged tracking distance of multiple cells over time. The EZ-Taxiscan experiments were independently performed six times and the data were largely consistent, although the extent of the toward-and-away motions of cells in 10−7 M S1P was variable depending on the experiment. Obvious away motion could clearly be observed in five of the six experiments (62 in 83 total cells), and the cells simply stopped in the middle of the chamber without clear backward migration in one of the six experiments (11 in 83 cells). (E) In vitro S1P-directed chemotaxis of RAW264.7 cells treated with siRNAs. Cells pretreated with control RNA duplex, siRNA against S1PR1, or siRNA against S1PR2 (Videos 4–6) were loaded into the EZ-Taxiscan chamber filled with a high concentration of S1P (10−9 M) in the other side. (F) RT-PCR detection of S1PR1 and S1PR2 in siRNA-pretreated RAW264.7 cells. Cells were pretreated with control RNA duplex (control siRNA), siRNA against S1PR1 (S1PR1 siRNA), or siRNA against S1PR2 (S1PR2 siRNA). (G) Migration distance data from microscopic analysis of control (red), S1PR1 knockdown (green), and S1PR2 knockdown (blue) cells. The experiments were independently performed three times and the data were largely consistent. Each dot represents the mean value of six independent cells and error bars represent SD.

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