Fig. 4
Roles of SOS2 in tumorigenesis and sensitivity to sulfarotene of HCC TRCs. a Higher levels of SOS2 protein were found in Hep3B-TRCs and PLC/PRF/5-TRCs than that in Hep3B, PLC/PRF/5 and L02 cells, as determined by western blotting. b Reduction of SOS2 protein levels in Hep3B-TRCs and PLC/PRF/5-TRCs by treatment with sulfarotene at 1.0 and 5.0 µM for 48 h, as revealed by western blotting. GAPDH served as the loading control. c Suppression of SOS2 levels by sulfarotene. Representative images for immunofluorescence staining of SOS2 (red) in Hep3B-TRCs and PLC/PRF/5-TRCs cultured in 3D soft fibrin gels after treatment with sulfarotene at 1.0 and 5.0 µM for 48 h. Blue, DAPI counterstain of the nucleus. d-e Effects of SOS2 overexpression on colony spheroid formation and sensitivity to sulfarotene of HCC TRCs. Hep3B-TRCs and PLC/PRF/5-TRCs stably overexpressing SOS2 were treated with sulfarotene at concentrations of 1.0 and 5.0 µM. After 48 h, the size of colony spheroids was determined in µm3. f-g Effects of SOS2 overexpression on xenograft tumor node formation and sensitivity to sulfarotene of HCC TRCs. Spheroids of PLC/PRF/5-TRCs stably overexpressing SOS2 were allowed to form in 3D soft fibrin gels for 1 week, and then inoculated subcutaneously into the flanks of nude mice. A bolus injection of sulfarotene at 0.22 or 2.2 mg/kg was given i.v. to nude mice once every two days. After 4 weeks, tumor nodes (e) were dissected from the mice and tumor volumes and weights (f) were measured (n = 6). Down arrow indicates the start of sulfarotene treatment. h Effects of SOS2 overexpression on the proliferation and apoptosis of tumor nodes and sensitivity to sulfarotene of HCC TRCs. Left, representative IHC images for the expression levels of SOS2, Ki-67 and Caspase-3 in sections of xenograft tumor nodes (e) from PLC/PRF/5-TRCs overexpressing SOS2 or after sulfarotene treatment compared to controls. Right, statistical analysis of the IHC scores in each group (n = 6). Tukey’s post hoc test. Data are presented as the mean ± SD of 3 independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant