Fig. 5

Effect of selenoprotein knock-down on TNFα stimulated NF-κB-driven reporter activity and interleukin 8 expression. a Semi-quantitative RTPCR amplification of mRNA levels for GPX1, SELW and SELH after knockdown of the respective gene with 80 pmol siRNA and in control cells. Total RNA was extracted, RTPCR carried out and the products separated by gel electrophoresis. b The intensity of bands corresponding to the amplified products was measured using UV Band software, normalised to the housekeeping gene GAPDH and expression then calculated relative to the cells treated with the negative control siRNA negative control. c Luciferase activity was measured in NF-κB-luc transfectant or TATA-luc transfectant Caco-2 cells grown in Se-supplemented medium but after GPX1, SELW or SELH siRNA knockdown and stimulation with 20 ng/ml TNFα for 4 h. Luciferase activity was measured in cell extracts, calculated per mg cell protein and expressed relative to the activity in the NF-κB-luciferase cells treated with negative control siRNA. *P < 0.05, **P < 0.01. d Cells were either stimulated with TNFα for 1 h or treated with carrier (phosphate-buffered saline). RNA was extracted and subjected to semi-quantitative RTPCR amplification of NF-κB target gene IL8 and housekeeping gene GAPDH. After separation of the amplified products by gel electrophoresis, intensity of bands was measured using UV Band software. IL8 mRNA levels were quantified, normalised against GAPDH and expression related to that in the cells treated with the negative control siRNA. *P < 0.05, **P < 0.01