Figure 2

Establishment of lenvatinib-resistant cells. (A) Lenvatinib-resistant Hep3B cells were established by treatment with lenvatinib for > 2 months (LR7.5-3B). Immunoblotting analysis of phosphorylated ERK (p-ERK), ERK, and ACTB levels in Hep3B and LR7.5-3B cells with or without 3 μM lenvatinib for 4 h. (B) Huh6 and JHH-4 cells were treated with 1.875 and 7.5 μM lenvatinib, respectively, for > 1 month. After treatment of the above-mentioned Huh6 and JHH-4 cells with 3 µM lenvatinib for 4 h, phosphorylation levels of ERK (p-ERK), ERK and TUBB were analyzed by immunoblotting. The intensity with the internal control were indicated. (C,D) Cell proliferation was analyzed by Cell Counting Kit 8 (CCK-8) assays in normal (Hep3B) and lenvatinib-resistant (LR7.5-3B) Hep3B cells that had been treated with 3 μM lenvatinib at the indicated times (n = 4 per treatment). Three independent experiments of (A–D) were performed, and representative results are shown. *p < 0.05.