Extended Data Fig. 10: Concurrent inhibition of ERK5 and ERK1/2 shows an enhanced anti-tumour effect in vitro.

a, Quantification of Fig. 3b with three independent experiments. The phosphorylation levels of each protein were normalized based on their total protein levels. n = 3 independent experiments. Data are mean ± s.e.m. P values were determined by two-sided unpaired Student’s t-test. b, Western blot analysis of EGFR–PAK2–ERK5 cascade in JHH1 cells, treated with lenvatinib, EGFR inhibitors (gefitinib and erlotinib) or their combination at the indicated concentrations for 6 h. HSP90 served as a loading control. c, The phosphorylation levels of each protein in b were normalized based on their total protein levels. n = 3 independent experiments. Data are mean ± s.e.m. P values were determined by two-sided unpaired Student’s t-test. d, EGFR expression of SNU449 cells was knocked down by two independent shRNAs, and cells were further treated with lenvatinib (5 μM) for 6 h. Western blot analysis of EGFR–PAK2–ERK5 cascade was performed. HSP90 served as a loading control. The pLKO vector was used in the control experiment. e, Quantification of Fig. 3c with three independent experiments. The phosphorylation levels of each protein were normalized based on their total protein levels. n = 3 independent experiments. Data are mean ± s.e.m. P values were determined by two-sided unpaired Student’s t-test. f, JHH1 cells were treated with lenvatinib (5 μM), PAK inhibitor FRAX1036 (2.5 μM) or their combination for 6 h, and western blot analysis was performed with the indicated antibodies. g, The phosphorylation levels of each protein in f were normalized based on their total protein levels. n = 3 independent experiments. Data are mean ± s.e.m. P values were determined by two-sided unpaired Student’s t-test. h, i, Long-term colony formation assays showing synergistic effects of lenvatinib and the PAK inhibitor FRAX1036 on the proliferation of SNU449 (h) and JHH1 (i) cells. Representative of three independent experiments. j, k, Long-term colony formation assays showing synergistic effects of lenvatinib and ERK5 inhibitor XMD8-92 on the proliferation of SNU449 (j) and JHH1 (k) cells. Representative of three independent experiments. l, m, SNU449 (l) and JHH1 (m) cells were treated with lenvatinib (5 μM), the ERK5 inhibitor XMD8-92 (2.5 μM) or their combination for 6 h, and western blot analysis was performed with the indicated antibodies. HSP90 served as a loading control. n–q, Knockout of ERK5 using the CRISPR–Cas9 system enhances the response to lenvatinib. The ERK5 knockout efficiency was determined by western blot in SNU449 (n) and JHH1 (p) cells. HSP90 served as a loading control. The effects of ERK5 knockout on proliferation were indicated by colony formation. The ERK5 knockout SNU449 (o) and JHH1 (q) cells, or their respective control cells were treated with DMSO or 5 μM lenvatinib. After 10 days of culture, cells were fixed, stained and photographed. NT, non-targeting sgRNA. r, Quantification of IHC staining in Fig. 3d. n = 6 per group. Data are mean ± s.e.m. P values were determined by two-sided unpaired Student’s t-test

Source data.