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Fig. 4 | Cancer Communications

Fig. 4

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

Fig. 4

Nuclear factor-κB (NF-κB) regulated RAP1 in an EYA4-dependent manner. a Western blotting analyses comparing the levels of RAP1 protein expression in Huh-7 and PLC5 cells after activation of β-catenin with epidermal growth factor (EGF, 100 ng/mL) or silencing of EYA4 with EYA4-targeted short hairpin RNA (shEYA4) in the presence or absence of pEYA4 transfection. b Chromatin immunoprecipitation (ChIP) analyses were performed to test the interactions between p65 and the RAP1A promoter in Huh-7 and PLC5 cells with or without pEYA4 transfection upon tumor necrosis factor-α (TNF-α, 10 ng/mL) stimulation. c Western blotting analyses comparing the levels of RAP1 protein expression in Huh-7 and PLC5 cells with TNF-α stimulation for the indicated times in the presence or absence of the NF-κB inhibitor BAY 11-7085 (5 µmol/L). d Western blotting analyses comparing the levels of RAP1 protein expression in Huh-7 and PLC5 cells upon TNF-α stimulation with or without p65-targeting small interfering RNA (p65 siRNA) transient transfection. e, f The expression of RAP1 protein (e) and RAP1A mRNA (f) was analyzed in Huh-7 and PLC5 cells transfected with pEYA4 alone or with pEYA4 plus shEYA4 in the presence or absence of TNF-α stimulation. **P < 0.01 vs. the pEYA4 group. g, h RAP1 and EYA4 protein expression (g) and RAP1A and EYA4 mRNA expression (h) in Huh-7 and PLC5 cells in which EYA4 was depleted by shEYA4 in the presence or absence of the NF-κB inhibitor BAY 11-7085. **P < 0.01 vs. shEYA4 group. i Western blotting analyses detecting the RAP1 protein abundance in Huh-7 and PLC5 cells in which EYA4 was silenced by shEYA4 transfection in the presence or absence of p65 siRNA transient transfection. The qRT-PCR experiments were performed five times with technical duplicates, and the real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ChIP chromatin immunoprecipitation, DMSO dimethyl sulfoxide, EGF epidermal growth factor, EYA4 eyes absent homolog 4, Flag-RAP1A Flag-tagged human RAP1A expressing plasmid, Flag-Vector Flag-tagged vector plasmid, GAPDH glyceraldehyde 3-phosphate dehydrogenase, IgG immunoglobulin G, IP immunoprecipitation, NF-κB nuclear factor-κB, P65 NF-κB p65 subunit, PCR polymerase chain reaction, pEYA4 EYA4-expressing plasmid, RAP1 RAS-related protein 1, Scr scramble short hairpin RNA, shEYA4 EYA4-targeted short hairpin RNA, siRNA small interfering RNA, TNF-α tumor necrosis factor-α

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