Fig. 6
CircUCK2(2,3) increases secreted TGFα and subsequent EGFR activation, and sensitizes HCC cells to EGFR inhibitors and the synergistic cytotoxic effect of lenvatinib combined with EGFR inhibitors. A and B ELISA assays of TGFα in PLC/PRF/5 with circUCK2(2,3) knockdown (A) or in SNU398 with WT or 149-mut circUCK2(2,3) overexpression (B). C Protein levels of pEGFR-(Y1068) and EGFR in HCC cells with circUCK2(2,3) knockdown or overexpression. D Schematic showing the procedure of medium transfer assay. Protein levels of pEGFR-(Y1068) and EGFR in SNNU398 cells treated with culture medium from SNU398 cells with WT or 149-mut circUCK2(2,3) overexpression. E Cytotoxic effects of pelitinib in SNU398 cells overexpressing WT or 149-mut circUCK2(2,3). F and G A bliss independent model to evaluate the synergistic killing of lenvatinib with pelitinib in SNU398 cells with or without circUCK2(2,3) overexpression in short-term viability assays and long-term clonogenic assays. Synergyfinder.org web tool was used to calculate the Bliss synergy scores. Average synergy scores for the indicated drug combinations were calculated. Statistical analyses were performed using paired Student’s t-tests (**p < 0.01; ***p < 0.001). siNC siRNA negative control, si-1 siRNA#1, si-2 siRNA#2, VC vector control, OE overexpression, 149-mut circUCK2(2,3) mutant with mutated miR-149-5p binding site, WT wild type, CM culture medium, IC50 half-maximal inhibitory concentration