Fig. 5

TGF-β enhances ID1/TNFAIP8 expression and inhibits apoptosis through a crosstalk between SMAD1 and NF-κB1. a A ChIP assay demonstrating SMAD1 antibody immunoprecipitates the NF-κB1 promoter in 8226R5 cells. H3 and igG antibodies were used as positive and negative control, respectively. b 8226-R5 cells were transfected with si-NF-κB1, si-SMAD1 or siRNA control (48 h), with or without TGF-β stimulation (6 h). The cell lysate was prepared and subjected to western blot with indicated antibodies. c The western blot analysis represents the indicated protein levels in RPMI-8226R5, MM1.R and OPM2 vel/R stimulated with TGF-β. d 8226-R5 and MM1.R cells were transfected with si-ID1 or siRNA control. The cell lysate was prepared 48 h after transfection and subjected to western blot with indicated antibodies. e Down-expression of SMAD1 alleviates TGF-β induced IKKε activation and IKBα degradation in 8226R5 cells. 8226R5 cells were transfected with si-NF-κB1, si-SMAD1 or siRNA control (48 h), with or without TGF-β stimulation (6 h). The cell lysate was prepared and subjected to western blot with indicated antibodies. f Cells were transiently co-transfected with reporter plasmids (pcDNA3.1 vector or TNFAIP8 plasmid) with siSMAD1 using liposome-3000 transfection reagent for 24 h. and then cell viability was evaluated by MTT assays. Results are presented as means±s.d. from at least three separate experiments. *: p < 0.05; **: p < 0.01; ***: p < 0.001; NS: not significant. g The cells were transiently co-transfected with reporter plasmids (pcDNA3.1 vector or TNFAIP8 plasmid) with siSMAD1 using liposome 3000 transfection reagent for 24 h, and then stained with annexin-V/propidium iodide and analyzed by flow cytometry to determine the percentage of apoptotic cells