Figure 3.

Effects of SphK2 overexpression on the sensitivity of HCC cells to regorafenib. (A) The protein expression of SphK2 in HCC cells transfected with LV-SphK2 lentivirus or control vector was measured by Western blot analysis. (B) Regorafenib sensitivity of HCC cells transfected with LV-SphK2 lentivirus or control lentivirus was assessed by the CCK-8 assay, and regorafenib IC₅₀ values were calculated accordingly. (C) The annexin V–FITC/propidium iodide double-staining assay, (D) cell cycle analysis, and (E) colony formation assay were applied to compare regorafenib sensitivity of HCC cells in the two different groups. (F) The expression levels of Bcl2, cleaved PARP, cyclin D1, CDK2, and CDK4 were examined by Western blot analysis. The dose of regorafenib treatment in the assays was 10 μM for 48 h, with the exception of the colony formation assay (5 μM, 14 days for SMCC-7721 and 10 days for MHCC-97H, respectively). The result is representative for three independent experiments. The error bars represent mean ± SD from a representative experiment. **p < 0.01, ***p < 0.001.