Figure 4.

SPHK1 induces the EMT by stimulating autophagy. (A) The inhibition of autophagy recovered the expression of epithelial markers and mesenchymal markers in SPHK1-overexpressing HCC cells. HepG2 cells stably expressing vector or MYC-SPHK1 were treated with autophagic inhibitors MHY1485 (2 μmol/L, 6 h), SBI-0206965 (10 μmol/L, 2 h), 3-MA (10 mmol/L, 6 h) and CQ (100 μmol/L, 12 h) and then harvested. EMT-related protein expression was detected by western blot analysis. (B) Suppression of autophagy reduced cell migration in SPHK1-overexpressing HCC cells. HepG2 cells stably expressing vector or MYC-SPHK1 were plated in the upper chamber of transwell filters for 24 h and then treated with 3-MA (10 mmol/L, 6 h) or CQ (100 μmol/L, 12 h). Then cells migrating to the underside of the transwell insert were measured. (C) Suppression of autophagy reduced the invasion of SPHK1-overexpressing cells. The transwell invasion assay was performed as described in (B), except that the chambers were coated with basement membrane Matrigel. (D, E) Statistical analysis of cells per field in (B) and (C). The cells per field were quantified as described in Materials and Methods. MHY, MHY1485; SBI, SBI-0206965; CQ, chloroquine.