Fig 2: S1PR3/PBX1 axis regulated cell cycle progress. (A) Extracellular and intracellular S1P contents were detected by S1P ELISA kit. **** p < 0.0001, extracellular versus intracellular, Student’s t-test. (B) RT-qPCR detected possible S1PRs expression in H460 cells after stimulation with exogenous S1P (5 µM) for 24 h. **** p < 0.0001, control versus S1P, Student’s t-test. (C) Western blot detected the expression of S1PR3, S1PR1 and SPHK1 in H460 shNC and shSPHK1 cells. (D) Western blot detected the expression of S1PR3, SPHK1, PBX1, CDK4, CDK2 and CyclinD1 in H460 cells with S1PR3 silence (siRNA 50 nM) in the presence or the absence of S1P (5 µM). (E) Flow cytometry analyzed cell cycle distribution in H460 cells with TY-52156 (5 µM) treatment in the presence or the absence of S1P (5 µM). (F) Gene correlation analysis between S1PR3 and PBX1 of LUAD in TIMER database. (G) Western blot detected the expression of p-Akt, Akt, S1PR3 and PBX1 in H460 cells with PBX1 silence or PBX1 overexpression. (H) RT-PCR was performed to determine gene abundance of S1PR3 promoter region in the different groups, which were immunoprecipitated using an anti-PBX1 antibody in H460 cells. **** p < 0.0001, Input versus IgG and IP versus IgG, Student’s t-test.

Fig 3: Hirudin suppresses TGF-ß-induced epithelial-mesenchymal transition via S1P/S1PR2/S1PR3 signaling-mediated PAR1 inhibition. (A) Expression levels of N-cad, Slug and E-cad in HK-2 cells were determined using western blotting. (B and C) a-SMA expression in HK-2 cells was analyzed by immunofluorescence analysis. Scale bar, 100 µm. Error bars represent the mean ± SEM from three independent experiments. ***P<0.001 vs. the control. ###P<0.001 vs. TGF-ß. ?P<0.05 and ???P<0.001 vs. TGF-ß+hirudun (1 mg/ml). S1PR, sphingosine-1-phosphate receptor; PAR1, protease-activated receptor 1; N-cad, N-cadherin; E-cad, E-cadherin; a-SMA, a-smooth muscle actin; S1P, sphingosine-1-phosphate; TFL, TFLLR-NH2.

Fig 4: Inhibition of S1PR2 and S1PR3 suppresses PAR1 expression. (A) Relative mRNA level of PAR1 in HK-2 cells was analyzed by reverse transcription-quantitative PCR. (B) PAR1 protein expression in HK-2 cells was quantified by western blotting. Error bars represent the mean ± SEM from three independent experiments. ***P<0.001 vs. the control. ##P<0.01 and ###P<0.001 vs. TGF-ß. S1PR, sphingosine-1-phosphate receptor; PAR1, protease-activated receptor 1.

Fig 5: S1PR3 suppresses the Hippo pathway in OS cells. a. A heat map showing the expression of the YAP target genes across MNNG-HOS NC samples and S1PR3 knockdown with shRNA samples. b and c. Relative mRNA levels of YAP target genes (CTGF and CYR61) in S1PR3-knockdown or sh-Control MNNG-HOS and U-2OS cells. Values are means ± SD, *p < .05, **p < .01, ***p < .001 (Student's t-test). d and e. Immunofluorescence of YAP in sh-Control and sh-S1PR3 MNNG-HOS and U-2OS cells. YAP is shown by red fluorescence, and the cell nuclei were stained with DAPI (blue fluorescence), Scale bars = 100 µm. f. Analysis of the percentage of cells with predominantly nuclear YAP. n = 5 randomly chosen fields. Values are means ± SD, ***p < .001 (Student's t-test). g. The level of phospho-YAP and total-YAP in sh-Control versus sh-S1PR3 MNNG-HOS and U-2OS cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)