Fig. 3: S1PR1 regulates ovarian cancer cell senescence through the PDK1-LATS1/2-YAP pathway.

A β-galactosidase staining was used to qualitatively (left) and quantitatively (right) analyze senescence changes in S1PR1 knockout A2780 and ES-2 cells. Scale bar, 50 µm. Values represent mean ± SD of three independent experiments. Student’s t-test; **p < 0.01. B q-PCR analysis of P27 and P53 expression in wild-type and S1PR1 knockout ovarian cancer cells. Values represent mean ± SD of three independent experiments. Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001. C Western blot showing the expression of P21, P27, P62, IGFBP7, PAI-1, histone H3, and p-H2AX in wild-type cells and S1PR1 knockout cells. GAPDH was used as control. D β-galactosidase staining was used to qualitatively (left) and quantitatively (right) analyze senescence changes in ES-2 cells after treatment with S1P1 receptor antagonist W146. Scale bar, 50 µm. Values represent mean ± SD of three independent experiments. One-way ANOVA; ***p < 0.001. E Western blot showing expression levels of PDK1, p-PDK1, LATS1, LATS2, YAP, and p-YAP in wild-type ovarian cancer cells and S1PR1-deficient ovarian cancer cells. GAPDH was used as control. F Western blot showing expression levels of PDK1, p-PDK1, LATS1, LATS2, PAI-1, and p-YAP after S1P treatment in ES-2 cells. GAPDH was used as control. G Immunofluorescence analysis of YAP expression and localization (green) following S1P treatment and no treatment. DAPI was used to counterstain nuclei (blue). Scale bar, 20 µm. H Western blot showing the expression of LATS1, LATS2, PDK1, p-PDK1, IGFBP7, and PAI-1 in ES-2 cells after treatment with PDK1 inhibitor BX517. GAPDH was used as control. I β-galactosidase staining analysis of senescence changes after BX517 treatment of ovarian cancer cells is shown qualitatively (left) and quantitatively (right). Scale bar, 50 µm. Values represent mean ± SD of three independent experiments. One-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001. J β-galactosidase staining analysis of WT cell senescence after BX517 (1 µM) and S1P (1 µM) treatment. Scale bar, 50 µm. Values represent mean ± SD of three independent experiments. One-way ANOVA; ns, p > 0.05, ***p < 0.001. K q-PCR analysis of LATS1/2 after S1P (1 µM) and BX517 (1 µM) treatment of ovarian cancer cells. Values represent mean ± SD of three independent experiments. One-way ANOVA; *p < 0.05, ***p < 0.001. L ELISA showing IL-6 levels in ES-2 cells treated with BX517 (2 µM) and W146 (100 µM) separately. Values represent mean ± SD of three independent experiments. One-way ANOVA; **p < 0.01, ***p < 0.001.