Figure 10: Inhibitory effects of K67 on tumour growth and tolerance to anti-cancer agents.

(a) Immunoprecipitation assays. Huh1 cells expressing FLAG-Keap1 were cultured in the presence or absence of indicated compound (50 μM) for 12 h. The immunoprecipitant with anti-FLAG antibody was subjected to immunoblot analysis. Experiments were performed three times. (b) Ubiquitination of Nrf2. Huh7 cells expressing S351E were cultured in the presence or absence of indicated compound (50 μM). After 4 h of treatment, the cells were treated with or without lactacystin (10 μM) for 8 h. The immunoprecipitant with anti-Nrf2 antibody was subjected to immunoblot analysis. Experiments were performed three times. (c) Immunoblot analysis. Huh1 cells were cultured in the presence of 50 μM K67 or Cpd16 for the indicated times. Cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis. Experiments were performed three times. (d) Quantification of mRNA levels of Nrf2 target genes in Huh1 cells treated with 50 μM K67 for the indicated times. Values were normalized to the amount of mRNA in non-treated Huh1 cells. Experiments were performed three times. Data represent means±s.e. *P<0.05, **P<0.01, ***P<0.001 as determined by the Welch t-test. (e) Effect of K67 on cell proliferation. Huh1 and Huh7 cells were pre-cultured in the presence of DMSO, K67 or Cpd16. Proliferation was measured starting at 72 h after pre-culture (n=4). Initial cell numbers were normalized to 1. Experiments were performed three times. Data represent means±s.e. *P<0.05, **P<0.01, ***P<0.001 as determined by the Welch t-test. (f) Effect of K67 on resistance to anti-cancer drugs. Huh1 and Huh7 cells were pre-cultured in the presence of DMSO or K67 for 96 h. Thereafter, the cells were treated with sorafenib, cisplatin or either drug in combination with K67 for 48 h, and survival ratio was determined (n=4). Experiments were performed three times. Data represent means±s.e. *P<0.05, **P<0.01, ***P<0.001 as determined by the Welch t-test. (g) Schematic diagram of cancer malignancy mediated by the p62–Keap1–Nrf2 axis.