Figure 3: The TGF-β-mediated inhibition of minimal LPL promoter activity in human macrophages is attenuated by transfection of plasmids specifying for DN forms of JNK, SEK-1 and c-Jun.
(A) Schematic representation of the regulatory region of the LPL gene identified in a previous study18. The −101 to +187 region linked to the luciferase reporter gene (Luc) is shown. The −31 to +187 sequence contains the TGF-β response elements (TGF-β RE) with three conserved Sp1/Sp3 binding sites required for the response (a single site at position +44 and a dual site at position +62/+65)18. The +9/+49 and +46/+90 sequences used for EMSA (Fig. 4) are also shown. (B) U937 cells were transfected with the minimal LPL promoter-luciferase construct (−101/+187 in the pGL2 Basic-luciferase vector) and DN JNK, DNSEK-1, DN c-Jun or pcDNA3 control vector. The cells were then differentiated with PMA (1 μM) for 12 h and then treated with vehicle (−, empty bars) or TGF-β (30 ng/ml) for further 12 h (+, filled bars). The luciferase activity was normalized to the protein concentration and is expressed as Relative Luciferase Activity. In each case, the value in cells treated with vehicle has been arbitrarily assigned as 100%. The data represent mean ± SD from three independent experiments. Statistical analysis was performed using the two-tailed unpaired Student’s t-test, *p < 0.05.
