Figure 5 | Oncogene

Figure 5

From: RMP promotes venous metastases of hepatocellular carcinoma through promoting IL-6 transcription

Figure 5

RMP upregulated IL-6 expression via interaction with p65 and RPB5. (a) Secretion of IL-6 by RMP in MHCCLM3 cells were completely inhibited by the NF-κB inhibitor (5 mM Bay11–7082). Data are mean±s.e.m. and are representative of three independent experiments. *P<0.05; ***P<0.001 (Student’s t-test). (b) Luciferase reporter assay of NF-κB was assessed in MHCCLM3 RMP, MHCCLM3-CMV, MHCCLM3-shRMP cells. Data are mean±s.e.m. and are representative of three independent experiments. *P<0.05; **P<0.01 (Student’s t-test). (c) MHCCLM3 cells co-transfected with pFlag-p65, pFlag-RMP, pGPU6-shRMP and pFlag-CMV. Data are mean±s.e.m. and are representative of three independent experiments. *P<0.05; **P<0.01; ***P<0.001 (Student’s t-test). (d) HEK293T cells were co-transfected with Flag-RMP, Flag-p65 and green fluorescent protein (GFP)-RPB5. At 48 h after transfection, cells were treated with tumor necrosis factor-α (TNFα) at a concentration of 10 ng/ml for 20 min before harvesting. Whole-cell lysates were immunoprecipitated with RMP, p65, GFP antibody respective or control immunoglobulin G and analyzed by western blotting. Cell lysates were also subjected to immunoblotting (lower panel). (e) HEK293T cells were transfected with small interfering RNA (siRNA) of RPB5 or control siRNA. At 12 h after transfection, Flag-RMP was transfected. 48 h later, cells were treated with TNFα at a concentration of 10 ng/ml for 20 min before harvesting. Whole-cell lysates were immunoprecipitated with Flag-tag antibody or control immunoglobulin G. Endogenous p65 was analyzed by western blotting. (f) A marked decrease of p65 bound on the upstream of transcriptional start site of IL-6 was found upon RMP knockdown MHCCLM3 and CSQT-2 cells as compared with the corresponding vector control in ChIP assay. Normal rabbit antibody added served as a negative control. Error bars represent s.e.m. from at least three independent experiments.*P<0.05; **P<0.01 (Student’s t-test).

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