Fig 2: TRAF4 deficiency impairs CHK1 activity and confers sensitivity to chemotherapy in CRC cells. a TRAF4 knockout reduces cell viability in HT29 cells with 5-Fu treatment. HT29 cells expressing sgCtrl or sgTRAF4 were treated with 5-Fu for 72 h and analyzed with the MTS assay. ***p < 0.001. b, c Anchorage-dependent (b) and -independent (c) growth of TRAF4-WT or TRAF4-knockout HT29 cells with 5-Fu treatment. ***p < 0.001. d TRAF4 knockout enhances 5-Fu-induced apoptosis. TRAF4-WT or TRAF4-knockout HT29 cells were treated with 5-Fu for 72 h, and WCEs were immunoblotted. e, f Anchorage-dependent (e) and -independent (f) growth of TRAF4-null HT29 cells rescued by exogenous WT TRAF4. ***p < 0.001. g-i Cell viability (g) and anchorage-dependent (h) and -independent (i) growth of CHK1 knockdown HT29 cells rescued by exogenous WT CHK1. ***p < 0.001. j–m TRAF4 knockout enhances the efficacy of 5-Fu in vivo. Tumor size was monitored (j). Tumors were dissected (k) and weighed (l). Ki67 and p-CHK1 were examined via IHC staining, and apoptosis was analyzed with the TUNEL assay (m). *p < 0.05, **p < 0.01. n–p TRAF4 reintroduction into TRAF4-null HT29 cells rescues tumorigenesis under 5-Fu treatment. TRAF4 WT or E3 ligase activity-deficient mutants were reintroduced into TRAF4-null HT29 cells and injected into NSG mice to establish the xenograft mouse model. Tumor size was monitored (n). Tumors were dissected (o) and weighed (p). **p < 0.01, ***p < 0.001. q CHK1 reintroduction into CHK1-knockdown HT29 cells rescues tumorigenesis under 5-Fu treatment. WT CHK1 or CHK1 K132R mutant was reintroduced into CHK1-knockdown HT29 cells and injected into NSG mice to establish the xenograft mouse model. Mice were treated with 5-Fu, and tumor size was monitored. ***p < 0.001

Fig 3: 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 ?-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

Fig 4: 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 5: CHK1 K132 was ubiquitinated by TRAF4. a Alignment of conserved CHK1 lysine residues among different species. Kinase, kinase domain; SQ, Ser/Gln cluster; CM, conserved motif. b Ubiquitination of CHK1 mutants by TRAF4 was determined by an in vivo ubiquitination assay in 293T cells transfected with various constructs. c Lysine (K) 132 is the ubiquitination site in CHK1. 293T cells were transfected with various constructs and subjected to in vivo ubiquitination analysis. d CHK1 phosphorylation in CHK1-null HT29 cells is rescued by exogenous WT CHK1. CHK1 WT or CHK1 knockout HT29 cells transfected with CHK1 WT or CHK1 K132R mutant were treated with 5-Fu and subjected to IB analysis. e In vitro kinase assay of WT and mutant CHK1. HT29 cells were transfected with various constructs and treated with 5-Fu. WCEs were immunoprecipitated with Flag-tag antibody. The IP proteins were incubated with CDC25C and subjected to an in vitro kinase assay. f In vivo ubiquitination of WT CHK1 and the S317/345 mutant by TRAF4 in 293T cells with 5-Fu treatment. g Chromatin-associated CHK1 ubiquitination in HT29 cells with 5-Fu treatment. TRAF4-WT or TRAF4-knockout HT29 cells were treated with 5-Fu for 24 h, and chromatin and non-chromatin fractions were isolated and subjected to immunoprecipitated and ubiquitination analysis. Chro, chromatin faction. Non-Chro, cytoplasmic proteins and soluble nuclear proteins. h Co-IP analysis of ATR and CHK1 interaction in chromatin fraction. TRAF4-WT or TRAF4-knockout HT29 cells were treated with 5-Fu for 24 h, and chromatin fraction was isolated and subjected to Co-IP analysis. i Chromatin-associated CHK1 WT and K132R mutant ubiquitination in HT29 cells with 5-Fu treatment. HT29 cells were transfected with Flag-CHK1 or Flag-CHK1 K132R and treated with 5-Fu for 24 h, and chromatin fraction was isolated and subjected to immunoprecipitated and ubiquitination analysis. j Co-IP analysis of ATR and CHK1 interaction in chromatin fraction. HT29 cells were transfected with Flag-CHK1 or Flag-CHK1 K132R and treated with 5-Fu for 24 h, and chromatin fraction was isolated and subjected to Co-IP analysis