Fig. 5

DDX27 enhanced and prolonged NF-кB signaling. a In left panel, different cancer pathway reporters consisting of specific pathway-focused transcription factor-responsive firefly luciferase construct were separately transfected to HCT116 cells along with renilla luciferase reporter as internal control. Right panel showed knockdown of DDX27 significantly inhibited NF-кB luciferase reporter activity in both HCT116 and SW480 cells. Reporter activity was determined as the ratio of firefly to Renilla luciferase activity. b Caffeic acid phenethyl ester (CAPE) or 4-methyl-1-N- (3-phenylpropyl) benzene-1,2-diamine (JSH-23) was added to cells at indicate concentration (For HCT116, 2.5 mg/L CAPE or 10 uM JSH-23 was used; For SW480, 10 mg/L CAPE or 20 uM JSH-23 was used). Control groups were treated with an equivalent dilution of DMSO. Cell viability was assessed by MTT assays. In both experimental groups (CAPE or JSH-23) and control groups (DMSO), the difference between growth rates of cells over-expressing DDX27 and cells carrying empty vector (EV) was determined by ANOVA with repeated-measures analysis of variances. c HCT116 cells stably overexpressing DDX27 and control cells were treated with 50 ng/ml TNF-α for 3 h. NFkB Signaling Pathway Plus PCR Array (Qiagen) which profiles the expression of 84 key genes related to NF-кB-mediated signal transduction was used to analyze the effect of DDX27 on the NF-кB signaling. The cutoff fold-change was set to 1.2. d and e SW480 or HCT116 cells were treated with 50 ng/ml TNF-α for indicated time. The mRNA expression levels of NF-кB target genes (BIRC3, CCL20, CXCL3, NFKBIA, TNF, and TNFAIP3) in SW480-EV and SW480-DDX27 (or HCT116-shCTL and HCT116-shDDX27) cells were measured by qPCR. Student’s t-test was performed between SW480-EV and SW480-DDX27 (or HCT116-shCTL and HCT116-shDDX27-2) cells at different time points. f SW480 or HCT116 cells stably overexpressing DDX27 and control cells were treated with 50 ng/ml TNF-α for indicated time. Isolated nucleus and cytoplasm were immunoblotted with indicated antibodies. Lamin A/C and α-tublin were used as nuclear marker and cytoplasmic marker, respectively. (*P < 0.05; **P < 0.01; ***P < 0.001)