FIGURE 4.

A20 blocks K13-induced up-regulation of CCL20 and IL-8 and cellular transformation. A–D, A20 blocks K13-induced up-regulation of CCL20 and IL-8. 293T cells were transfected with an empty vector, K13, or K13-ER^TAM along with luciferase reporter constructs driven by CCL20 (CCL20-Luc) or IL-8 promoters (IL-8-Luc) (75 ng/well) and a pRSV/LacZ (β-galactosidase) reporter construct (75 ng/well) in the presence or absence of A20. The cells transfected with K13-ER^TAM were subsequently treated with 4OHT (20 nm) for 24 h and the luciferase reporter assay performed essentially as described in Fig. 2A. Lower panels demonstrate the expression of the transfected proteins as measured by immunoblotting with the indicated antibodies. The values shown are the averages (mean ± S.E.) of one representative experiment of three in which each transfection was performed in duplicate. E and F, cellular supernatants from 293T cells transfected, as explained in A–D, were collected before doing luciferase assay and used to measure the secretion of CCL20 and IL-8 by ELISA. The values shown are averages (mean ± S.E.) of one representative experiment of three in which the level of CCL20 or IL-8 secretion was measured in duplicate. Asterisks (*) indicate significance at levels of p ≤ 0.05. G, immunoblot shows increased A20 expression in Rat1-K13 cells transfected with an A20 expression construct. H, soft agar colony formation by Rat1-K13 cells transfected with an A20 expression vector was reduced compared with those transfected with an empty vector. The values shown are mean ± S.D. of two independent experiments performed in triplicate. *, p ≤ 0.05 versus vector cells.