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. 2022 Dec 21;13(12):1065. doi: 10.1038/s41419-022-05511-3

Fig. 6. Inhibition of SPHK1/S1PR3 signaling triggers autophagic death in TSC2-deficient cells.

Fig. 6

A Gene expression of ATG5 in 621-101 cells treated with vehicle control or specific SPHK1 inhibitor PF543 (2.5 μM) was detected by RT-qPCR. 621-101 cells. B 621-101 cells were transfected with shRNA-SPHK1 or negative control (shN.C). Immunoblotting was performed to determine the levels of phospho-S6 (235/236), LC3I and LC3II in 621-101 shRNA-SPHK1 cells and control cells treated with vehicle control, rapamycin (Rapa, 10 nM) or CQ (5 µM). C 621-101 cells were treated with 20 nM rapamycin and 5 µM CQ, with or without 2.5 µM PF543 for 24 h. Levels of tuberin, p62, phospho-S6 (235/236), LC3I/LC3II, cl-caspase 3 and SPHK1 were assessed by immunoblotting. 621-103 cells were added as TSC2-addback control. D, E 621-101 cells were transfected with S1PR3-siRNA or negative control (siControl). Gene expression of ATG5 and SQSTM1 was analyzed by RT-qPCR (D), and protein levels of p62, LC3I/LC3II were determined by immunoblotting (E). F 621-101 cells were treated with vehicle control or selective S1PR3 inhibitor TY52156 (2 μM), and the gene expression of ATG5, ATG7, ATG12, BECN1 and SQSTM1 was detected by RT-qPCR. G 621-101 cells were treated with 20 nM Rapa and 5 µM CQ with or without 2 µM TY52156 for 24 h. Protein levels of tuberin, p62, phospho-S6 (235/236), LC3I/LC3II and S1PR3 were assessed by immunoblotting. 621-103 cells were added as TSC2-addback control. Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001.