Fig. 3: NF1 and DUSP9 loss induce lenvatinib resistance.

A Changes in NF1 and DUSP9 expression upon shRNA knockdown were analyzed by Western blotting. NF1 shRNA2 and DUSP9 shRNA1 have the best interference effect, and can be used as NF1 and DUSP9 knockdown interference cell line for further experiments. B NF1 and DUSP9 knockout expression levels were confirmed by western blotting in PLC/PRF/5 cell. Expression of NF1 or DUSP9 is undetectable in PLC/PRF/5 cell after knocked out by NF1 or DUSP9 sgRNA, respectively. C KRAS(G12V) overexpression levels were analyzed by anti-Flag blots (WB) in Huh7 and PLC/PRF/5 cells. DNA from SW480 cell line (KRAS G12V mutation) served as a positive control. D Comparison of IC50 based on MTT assay in Huh7 and PLC/PRF/5 cells. IC50 was significantly increased after NF1 and DUSP9 were knocked out or knocked down compared with those in NC. E Clone formation assay in t Huh7 and PLC/PRF/5 cells using gene knockout or knockdown assays. All NF1 and DUSP9 knockout or knockdown clones showed markedly enhanced colony formation capacities from those in control cells. F NF1 and DUSP9 overexpression levels were confirmed by western blotting in Huh7 and PLC/PRF/5 cells. The repression of NF1 and DUSP9 by sgRNA knocked out could be reversed after NF1 and DUSP9 overexpression. G MTT assay showed that IC50 value after NF1 and DUSP9 overexpression was significantly decreased again compared with those in NF1 and DUSP9 knockout or knockdown cells. H NF1 and DUSP9 overexpression reversed the colony formation capacities enhanced by NF1 and DUSP9 loss. Error bars represent mean ± SEM for triplicate experiments, *P < 0.05, **P < 0.01, ***P < 0.001, ns no significantly changed.