Panel A - qRT-PCR for S1PR1 in RH30 cells transfected with plasmids
encoding shRNA against Renilla (control) and against S1PR1. The experiment was repeated
twice on three different batches of cells with similar results. **p < 0.01.
Panel B – FACS analysis of S1PR1 expression in RH30-shRenilla and
RH30-shS1PR1 cells. The experiment was repeated three times with similar results.
**p < 0.01. Panel C - Chemotaxis of RH30 cells transfected with
plasmid encoding shRNA against Renilla (control) and against S1PR1 across Transwell
membranes in response to S1P (1 μM). The results from three independent experiments are
shown as means ± SD. *p < 0.05 or **p < 0.01 compared with the
control. Panel D - RH30 tumor formation after downregulating S1PR1
expression. Tumor formation by RH30-shRenilla (control), and RH30-shS1PR1 cells
inoculated into the hind limb muscles of SCID/Beige inbred mice. Five weeks later, mice were
sacrificed and femora harvested to evaluate the size of the growing tumor.
**p<0.01 compared with the control. Panel E - Detection of human RMS
cells in bone marrow (BM), liver, lung, and peripheral blood (PB) by qRT-PCR. The results are shown
as means ± SD. *p < 0.05 or **p < 0.01.