Fig 2: Ubiquitination of SphK2 suppresses glioma cell progression in vitro. (A) The expression levels of SphK2 and NEDD4L in normal brain cells and glioma cells. (B–D) Glioma U251 cells were transfected with NEDD4L and/or SphK2 overexpression plasmid or transfected with NEDD4L and/or SphK2 siRNA, and cell viability, apoptosis, and invasion were measured by the CCK8 assay, invasion assay, and flow cytometry assay in sequence. *P < 0.05, **P < 0.01 compared with the vector or NC; #P < 0.05, ##P < 0.01 compared with SphK2 overexpression plasmid or siRNA.

Fig 3: Ubiquitination of SphK2 inhibits glioma growth in vivo. (A,B) Glioma U251 cells stably overexpressed of NEDD4L and/or SphK2 were implanted into 5-week-old female nude mice, and tumor weight was measured 21 days after injection. *P < 0.05, **P < 0.01 compared with the vector; ##P < 0.01 compared with SphK2 overexpression plasmid.

Fig 4: NEDD4L interacts with SphK2. (A) Anti-SphK2 was used to precipitate the protein complexes in HEK293T cells and anti-NEDD4L was used to perform co-IP assay. (B) HEK293T cells were transfected with SphK2-Flag and/or NEDD4L-HA overexpression plasmid. Anti-Flag was used to precipitate the protein complexes and anti-HA was used to perform the co-IP assay. (C) Fluorescence co-localization assay was used to confirm that NEDD4L was co-expressed with SphK2 in the cytoplasm in HEK293T cells.

Fig 5: SphK2 deficiency downregulates CERT both in vivo and in vitro.A, B Wild-type (WT) and Sphk2 knockout (KO) mice were fed with a high-fat, high-sugar diet (HFHSD) for 46 weeks. A Proteins were extracted from non-tumorous liver tissues and analyzed using Western blotting. Expression levels of the indicated proteins were quantified; n = 8. B mRNA expression of Cert1, the gene encoding mouse CERT, was examined by real-time RT-PCR; n = 8. C–E Huh7 hepatic cells were transduced with lentiviral-based short hairpin RNA (shRNA) to knock down SphK2 and then treated with a combination of free fatty acids (FFA, 200 µM palmitate + 400 µM oleate) for 48 h. C, E Proteins were extracted from cells and analyzed using Western blotting. The CERT protein expression and phospho-p65 over total p65 (p-p65/t-p65) were quantified; n = 5. D mRNA expression of CERT1, the gene encoding human CERT, was examined in the presence and absence of the FFA treatment by real-time RT-PCR; n = 4. A–E Data are expressed as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; versus control. F The correlation of SPHK2 and CERT1 mRNA expression was analyzed in human HCC subjects with body mass index > 25. Data are sourced from TCGA (https://portal.gdc.cancer.gov).