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Fig. 5 | Cancer Communications

Fig. 5

From: EYA4 inhibits hepatocellular carcinoma growth and invasion by suppressing NF-κB-dependent RAP1 transactivation

Fig. 5

The serine/threonine-phosphatase activity of EYA4 inactivated NF-κB signaling. a, b Western blotting analyses examining the levels of IκBα phosphorylation and p65 nuclear translocation in Huh-7 (a) and PLC5 (b) cells transduced with pEYA4 in the presence or absence of the serine/threonine phosphatase inhibitor calyculin A (CA; 10 nmol/L) upon TNF-α (10 ng/mL) stimulation. Histone H2.AX served as the loading control for the nuclear fractions. c Representative immunofluorescence staining images comparing the nuclear translocation of p65 in Huh-7 and PLC5 cells transfected with pEYA4 in the presence or absence of CA treatment upon TNF-α stimulation. Scale bar = 20 µm. d, e In vivo ubiquitination assays comparing the polyubiquitination levels of IκBα in Huh-7 (d) and PLC5 (e) cells transfected with pEYA4 in the presence or absence of CA treatment upon TNF-α stimulation. f EYA4 opposed the transcriptional programs of NF-κB signaling in HCC cells. Heat map of gene expression microarray data showing the expression of NF-κB signaling-dependent genes in the indicated HCC cells. g, h qRT-PCR analyses of the NF-κB downstream target genes B cell leukemia/lymphoma 2 (BCL-2), cyclin D1 (CCND1), twist-related protein 1 (TWIST1), and X-linked inhibitor of apoptosis (XIAP) in Huh-7 (g) and PLC5 (h) cells transfected with pEYA4 in the presence or absence of CA upon TNF-α stimulation. The experiments were performed five times, each qRT-PCR assay was performed in technical duplicates, and the real-time values were normalized to GAPDH. *P < 0.05 versus the TNF-α plus pEYA4 groups. BCL-2 B cell leukemia/lymphoma 2, CA calyculin A, CCND1 cyclin D1, DAPI 4′,6-diamidino-2-phenylindole, EYA4 eyes absent homolog 4, GAPDH glyceraldehyde 3-phosphate dehydrogenase, IκBα nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, IP immunoprecipitation, NF-κB nuclear factor-κB, P65 NF-κB p65 subunit, p-IκBα phosphorylated IκBα, p-P65 phosphorylated P65, PCR polymerase chain reaction, pEYA4 EYA4-expressing plasmid, RAP1 RAS-related protein 1, shEYA4 EYA4-targeted short hairpin RNA, siRNA small interfering RNA, TNF-α tumor necrosis factor-α, TWIST1 twist-related protein 1, Ub ubiquitination, (Ub) n polyubiquitination, WB Western blotting, XIAP X-linked inhibitor of apoptosis

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