Fig. 7
Arg319 of DDX39B is required for PKM2 binding and the promotion of CRC carcinogenesis and metastasis. a A Ni-NTA pull-down assay was carried out using bacterially-expressed wild type and the indicated mutant forms of GST-tagged DDX39B and His-tagged PKM2 in vitro. b The binding interface between DDX39B and PKM2 was based on the molecular docking model. c–f, h–k The indicated CRC cells were divided into three groups: stably expressing mock, DDX39BWT, and DDX39BR319A. c Interaction between PKM2 with DDX39BWT or DDX39BR319A was determined by immunoprecipitation assay. d Cell lysates were immunoprecipitated with anti-PKM2 antibody, and the ubiquitination of PKM2 was detected. e The phosphorylation of PKM2S37 was determined by western blotting. f Binding of PKM2 with importin α5 was examined by immunoprecipitation assay. g HCT116 and SW620 cells were transfected with the indicated plasmids, and subcellular localization of DDX39B and PKM2 signals were observed by immunofluorescence assay. h The phosphorylation of STAT3Y705 and histone H3T11 were detected by western blotting. (i) The relative transcription of c-Myc, GLUT1, LDHA, Cyclin D1 and MEK5 genes was measured by qPCR. The indicated CRC cells were orthotopically inoculated into the cecum of mice (n = 6). At day 60 after inoculation, the bioluminescent images and light emission of orthotopic tumors were captured and quantified (j). The representative bioluminescent images of the isolated lungs and livers were obtained, and the metastases were quantified (k). Data are presented as mean ± SD. The p values were obtained by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001
