Fig. 2

CircRNA-SORE is critical for maintaining sorafenib resistance. a Relative cell viability of three HCC sorafenib-resistant cell lines with circRNA-SORE siRNA knockdown (si-circRNA-SORE) compared with controls treated with the indicated concentrations of sorafenib (7 μM for HepG2-SR, 6 μM for LM3-SR and SKhep1-SR) for 72 h. b Colony formation assays in three HCC sorafenib-resistant cell lines with or without shcircRNA-SORE. Cells were all treated with sorafenib (7 μM for HepG2-SR, 6 μM for LM3-SR and SKhep1-SR) for 24 h in complete media, washed with PBS and cultured in complete media for another 7 days. c HepG2-P and HepG2-SR cell lines transfected with si-circRNA-SORE or negative control siRNA (si-NC) were treated with sorafenib. Cell viability was recorded by Real-time Cell Analysis xCELLigence System and three technical repetitions were performed. ΔAUC (difference of area under curve) = (AUC NC) – (AUC si-SORE). Each technical repetition yields a ΔAUC, and t tests were performed. d Similar to (c) except no sorafenib was added. e Brightfield images (objective magnification = 10) showing cell morphology of three sorafenib-resistant cell lines with or without si-circRNA-SORE or sorafenib at the indicated concentrations (7 μM for HepG2-SR, 6 μM for LM3-SR and SKhep1-SR) for 72 h. Scale bar, 20 µm. f Western blot analysis of sorafenib-resistant cell lines transfected with si-circRNA-SORE using PARP antibody (upper) and β-tubulin antibody (lower). g PI/Annexin V flow cytometry analysis (10,000 cells) of sorafenib-resistant cells transfected with si-NC or si-circRNA-SORE. h TUNEL staining (green) of three sorafenib-resistant cell lines transfected with si-NC or si-circRNA-SORE. Nuclei were stained with DAPI (blue). Scale bar, 20 µm. i Kaplan−Meier recurrence-free survival analysis for sorafenib-treated HCC patients with low and high circRNA-SORE expression. The median circRNA-SORE expression was used as cut-off for high- and low-expression groups. Three independent experiments with three technical repetitions were performed. Data are expressed as mean ± SEM (error bars). Statistical analyses used Student’s t test and Kaplan−Meier survival analysis. *p < 0.05, and **p < 0.01