Figure 5.

MT1G suppresses NF-κB -activin A pathway through promoting TRAF7-mediated degradation of p65. (A) IB analysis of NF-κB p65 subunit phosphorylation in MT1G overexpressing (MT1G-GFP) and control (GFP) BxPC-3-Gem cells. (B) Luciferase reporter assay in MT1G overexpressing and control BxPC-3-Gem cells post gemcitabine treatment for 48 hours. RLU, relative luminescence units. (C) IB analysis of NF-κB p65 subunit phosphorylation in BxPC-3 and BxPC-3-Gem cells. (D) Luciferase reporter assay to determine the activation of NF-κB in the indicated cells treated with gemcitabine for 48 hours. (E, F) ELISA analysis of the secreted activin A protein levels in indicated GFP or MT1G-GFP overexpressing BxPC-3-Gem (E) or PANC-1 (F) cells transfected with Flag empty vector (Flag-EV) or Flag-tagged p65 plasmid (Flag-p65) for 48 hours. (G, H) IB analysis of NF-κB p65 subunit in MT1G overexpressing and control BxPC-3-Gem cells (G), or BxPC-3 and BxPC-3-Gem cells (H) treated with cycloheximide (CHX) (100 μg/mL) for up to 24 hours (left). Quantification of p65 was normalized to the loading control and expressed relative to 0 hour (G bottom, H right). (I) PANC-1 cells were co-transfected with indicated plasmids for 48 hours and treated with MG132 (1 μM) for 12 hours and subjected to immunoprecipitation (IP) with the anti-Flag antibody, followed by IB with anti-HA and anti-Flag antibodies. Whole cell expression (input) of proteins were detected by IB with indicated antibodies. (J-L) RT-qPCR analysis of TRAF7 mRNA levels in BxPC-3 and BxPC-3-Gem cells (J), MT1G knockdown BxPC-3 cells (K) or MT1G overexpressing BxPC-3-Gem cells (L). RE, relative expression. (M) MT1G overexpressing and control BxPC-3-Gem cells were transfected with control siRNA (si-NC) or siRNA targeting TRAF7 (si-TRAF7) for 48 hours and subjected to IB with indicated antibodies. Data are presented as mean ± SD (n = 3) and are representative of at least three independent experiments, *P < 0.05, **P < 0.01 by two-tailed Student's t test.