Fig. 7

CHK1 inhibitor overcomes chemoresistance in vitro and in vivo. a IB analysis of TRAF4 and p-CHK1 in parental and chemoresistant CRC cells. The cell lysates of parental SW620, HT290, and HCT8 cells and 5-Fu-resistant CRC cells were treated with 5-Fu for 24 h, and WCEs were subjected to IB analysis. *5-Fu-resistant clones. b Correlation between TRAF4 and p-CHK1 in parental cells and 5-Fu-resistant clones displayed in a. Pearson’s coefficient tests were performed to assess statistical significance. c TRAF4 knockout enhances the sensitivity of HT29 cells to 5-Fu. HT29 parental cells and 5-Fu-resistant (HT29R) cells were transfected with control or TRAF4 sgRNA and treated with 100 μM 5-Fu for 72 h, followed by MTS analysis. ***p < 0.001. d, TRAF4 knockout reduces colony formation in HT29 cells treated with 5-Fu. The stable cells generated in c were treated with 100 μM 5-Fu for 24 h and subjected to a plate colony formation assay. ***p < 0.001. e, IB analysis of apoptosis-related proteins in HT29R stable cells treated with 5-Fu. f-i Cell viability (f), plate colony formation assay (g), soft-agar assay (h), and IF detection of γ-H2AX (i) in HT29R cells treated with the vehicle control, 100 μM 5-Fu, the CHK1 inhibitor, prexasertib, or a 5-Fu + prexasertib combination. ***p < 0.001. j IB analysis of apoptosis-related proteins in HT29R and SW620R cells treated with the vehicle control, 5-Fu, prexasertib, or a 5-Fu + prexasertib combination. k, l Tumorigenesis of HT29R cells treated with 5-Fu and prexasertib. Tumor size was monitored (k), and tumors were weighed (l). *p < 0.05, **p < 0.01, ***p < 0.001. m A working model of TRAF4-mediated CHK1 ubiquitination and activation in cancer cell chemotherapy resistance