Figure 6.

Hypoxia and S1P stimulate YAP1 nuclear translocation and verteporfin attenuates S1P-mediated YAP1 nuclear translocation in PASMCs. Exposing hPASMCs to hypoxia (3%, 30 min) significantly increased YAP1 translocation to the nucleus as determined by immunofluorescence (A), and nuclear YAP1 expression levels were quantified and are shown in (B). Similarly, the S1P (0.1 µM and 1 µM) treatment of PASMCs for 30 min induced YAP1 nuclear translocation, an effect that was abrogated by the pretreatment of cells with verteporfin (100 nM for 1 h), followed by an S1P challenge for 30 min (C,D). hPASMCs were pretreated with the S1PR2 antagonist JTE-013 (2 µM) for 1 h followed by S1P (10-1000 nM) challenge for 24 h. YAP1 and TAZ expressions were determined by Western blotting, and a representative blot is shown (E) and quantified by image analysis, and it is normalized to the total actin for YAP1 (F) and TAZ (G). hPASMCs were transfected with adeno control vector or SPHK1 dominant mutant (5 MOI, 24 h) prior to exposure to normoxia or hypoxia (3%, 2 h). Cells were subjected to the isolation of their nuclei using a commercial kit; subjected to Western blotting; and probed with anti-YAP1, anti-Lamin B, and anti-SPHK1 antibodies. The baseline and hypoxia-induced nuclear localization of YAP1 were reduced in hPASMCs transfected with SPHK1DN adenoviral plasmid compared with cells transfected with a control plasmid (H,I). n = 3–5. * p < 0.05 versus normoxia or vehicle control; ** p < 0.01 versus vehicle control; *** p < 0.05 versus S1P; # p < 0.05 versus vector control. Scale bar: 25 μm.