FIGURE 6.

CRNDE/ATG4B/autophagy axis attenuates the sensitivity of sorafenib in HCC cells. (A–C) HepG2 and Hep3B cells were treated with various concentrations of sorafenib for 24 h. Then the levels of CRNDE and ATG4B mRNA were determined by qPCR (A,B), and the levels of SQSTM1/p62, LC3, and ATG4B protein were measured by Western blot (C). (D,E) HepG2 and Hep3B cells were transfected with si-CRNDE, si-ATG4B (or si-NC), or treated with 20 μM chloroquine (CQ) (or vehicle control DMSO) for 12 h, and then treated with 10 μM sorafenib for 24 h. Subsequently, the cell viability was analyzed by CCK-8 assay (D), and the levels of cleaved PARP, SQSTM1/p62, LC3, and ATG4B were tested by Western blot (E). (F) After treatment as in panels (D,E), the cells were stained with Hoechst 33258, and then observed under a fluorescence microscope (scale bar: 20 μm; white arrows indicate the apoptotic cells). (G) After treatment as in panels (D,E), the cells were stained with annexin V-FITC/PI, and then the cell apoptosis was assayed by flow cytometry. (H) After co-transfected with si-CRNDE (or si-NC) and pCMV-ATG4B (or pCMV) for 12 h, HepG2 and Hep3B cells were treated with 10 μM sorafenib for 24 h, and then the cell viability was detected by CCK-8 assay. (I) HepG2 and Hep3B cells were transfected with si-CRNDE, si-ATG4B (or si-NC) for 12 h, followed by the co-treatment with 0.1 μM rapamycin (RAPA) and 10 μM sorafenib (or vehicle control) for 24 h. Subsequently, the cell viability was examined by CCK-8 assay. si-CRNDE: the siRNA for CRNDE; si-ATG4B, si-NC, pCMV-ATG4B and pCMV were the same as the description in Figure 2; ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001.