Fig. 2: SPHK1 mediates tumorigenesis of TSC2-deficient cells in vitro and in vivo.

A LAM patient-derived TSC2-deficient (621-101) cells were transfected with shRNA-SPHK1 (#1 and #2) or a negative control (shNC). The mRNA and protein levels of SPHK1 were determined by RT-qPCR and immunoblotting, respectively. B Viability of 621-101shNC and 621-101 shSPHK1 cells cultured in serum-free medium for 24 h was measured by MTT assay (upper panel). The cell death rate was measured by PI/CV assay (lower panel). The results are representative of eight independent samples per group. C 621-101shNC and 621-101 shSPHK1 cells were seeded in 6-well plates for 24 h and then stained with an Annexin V:FITC apoptosis detection kit. Cell death was analyzed by flow cytometry. D The capacities for 621-101shNC and 621-101 shSPHK1 cell migration (upper panel) and invasion (lower panel) were determined in a Transwell chamber without or with Matrigel. Cells were cultured in serum-free medium for 48 h and stained with crystal violet. Representative images of three repeats in each group. E 621-101shNC and 621-101 shSPHK1 cells transfected with firefly luciferase were injected intravenously into SCID mice (n = 5). Lung colonization was determined by bioluminescence imaging 0 h, 2 h, 6 h, and 12 h post injection. Luminescence color scale: 0 h, 2 h, 6 h, and 12 h (min = 1 × 10⁶, max = 1 × 10⁷). The statistical analysis (lower panel) indicated the relative photon flux change. The results are representative of five mice per group. Student’s t test, **P < 0.01, ***P < 0.001, and ****P < 0.0001.