Fig 2: CHK1 interacts with TRAF4. a Scatterplot showing the proteins pulled down by CHK1 and Control IgG antibodies. b Co-immunoprecipitation (Co-IP) analysis of the TRAF4 and CHK1 interaction in 293T cells transfected with the indicated constructs. WCE, whole-cell extract. c Co-IP analysis of the endogenous TRAF4-CHK1 interaction in HT29 cells. d Cell-free binding between TRAF4 and CHK1. e, f Schematic of CHK1 WT and deletion mutants (e) and TRAF4 WT and deletion mutants (f) used in this study. N, N-terminal; C, C-terminal; RING, RING domain; Intermediate, zinc finger and coiled-coil domain; TRAF, TRAF-C domain. g Co-IP analysis of TRAF4 and CHK1 truncation mutant interactions. 293T cells were transfected with the indicated constructs and subjected to Co-IP and immunoblotting (IB) analysis. h Co-IP analysis of CHK1 and TRAF4 truncation mutant interactions. 293T cells were transfected with the indicated constructs and subjected to Co-IP and IB analysis. *Non-specific signal

Fig 3: CHK1 phosphorylation positively correlates with TRAF4 overexpression in CRC patient samples. a Representative images of IHC staining for Ki67, TRAF4, p-CHK1 (S345), and CHK1 from 92 cases of primary tumors with matched adjacent tissues. b, c IHC scores for TRAF4 (b) and p-CHK1 (c) in 92 paired tumors and adjacent tissues. ***p < 0.001. d Kaplan–Meier curve shows the relationship between overall survival and TRAF4 expression in CRC patients. Data were obtained from the following online tool: https://hgserver1.amc.nl/cgi-bin/r2/main.cgi. e Representative images of IHC staining from a CRC primary tumor with high TRAF4 and p-CHK1 (S345) protein levels of or low TRAF4 and p-CHK1 (S345) protein levels. f The expression of TRAF4 and p-CHK1 (S345) is positively correlated. Pearson’s coefficient tests were performed to assess statistical significance. g The percentage of tissues displaying low or high TRAF4 and p-CHK1 (S345). ***p < 0.001. h Representative images of relapse tissues analyzed by IHC staining for Ki67, TRAF4, p-CHK1 (S345), and CHK1. i, j IHC scores of TRAF4 (i) and p-CHK1 (S345) (j) in paired primary and relapse samples. ***p < 0.001. k The expression of TRAF4 and p-CHK1 (S345) positively correlates in relapsed CRC tissues. Pearson’s coefficient tests were performed to assess statistical significance. l The percentage of specimens at initial diagnosis (left) and relapse (right) that display low or high TRAF4 and p-CHK1 (S345) levels after combination treatment. ***p < 0.001

Fig 4: TRAF4 promotes K63-linked ubiquitination of CHK1 is required for CHK1 activation. a In vivo CHK1 ubiquitination by TRAF4 in 293T cells transfected with various constructs. b CHK1 ubiquitination by TRAF4 is K63-linked. An in vivo ubiquitination assay was performed in 293T cells transfected with various constructs and treated with 5-Fu. c In vivo CHK1 ubiquitination by TRAF4 mutants. TRAF4 WT, TRAF4 (DM-RING) mutant, or TRAF4 (C18A) mutant were co-transfected into 293T cells with various constructs, and WCEs were subjected to an in vivo ubiquitination assay. d In vitro CHK1 ubiquitination by TRAF4. e Endogenous CHK1 ubiquitination by TRAF4 in HT29 cells. TRAF4-WT or TRAF4-knockout HT29 cells were treated with 5-Fu for 24 h, and WCEs were immunoprecipitated and subjected to ubiquitination analysis. f CHK1 ubiquitination in TRAF4-null cells is rescued by exogenous WT TRAF4. TRAF4-knockout HT29 cells were transfected with various constructs and treated with 5-Fu, and WCEs were immunoprecipitated for IB. g CHK1 phosphorylation in TRAF4-null cells is rescued by exogenous WT TRAF4. TRAF4-knockout HT29 cells were transfected with various constructs and treated with different doses of 5-Fu, and WCEs were immunoblotted. *Non-specific signal

Fig 5: 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