Figure 5

Real-time RT-PCR, quantitative RT-PCR, transient transfection assays, western blotting and cell growth of HEC-50B and KLE cells after transfection with the emmprin siRNA. A) Real-time PCR of TGF-β and EGF expression after transfection of the emmprin siRNA into HEC-50B and KLE cells for 72 h. B) Quantitative PCR analyses of E-cadherin, Vimentin and Snail expression levels after transfection of the emmprin siRNA into HEC-50B and KLE cells for 72 h. GAPDH was used as a loading control. C) Transient transfection assays of NF-κB and AP-1 into HEC-50B and KLE cells after transfection with the emmprin siRNA for 72 h. The assays were carried out for quadruplicate transfection experiments. D) Western blot analysis of NF-κB p65 and p-p65 expression after transfection of the emmprin siRNA into HEC-50B and KLE cells for 72 h. An anti-β-actin antibody was used as a loading control in the same membrane. E) PCR analyses of VEGF, MMP-2 and MMP-9 expression levels after transfection of the emmprin siRNA into HEC-50B and KLE cells for 72 h. GAPDH was used as a loading control. F) Cell growth in monolayers after transfection of the emmprin siRNA. The monolayer growth was evaluated for HEC-50B and KLE cells transfected with the emmprin siRNA and cultured for 1, 3, 5 and 7 days in DMEM or DMEM/F12 medium supplemented with 10% FBS. The numbers represent the data from triplicate experiments.