Fig. 5

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