Figure 3

Blocking endogenous ADAM17 inhibits IL-13-induced effects. Antisense oligonucleotides directed against ADAM17 (antisense), or corresponding scrambled oligonucleotides (scrambled), were added to NHBE cell cultures for 2 days. Cultures containing no oligonucleotides received the transfection reagent (FuGene6) during this time. On the third day, cells were exposed to control media, IL-13 (10 ng/ml), or TGFα (5 ng/ml), with or without the addition of the scrambled or antisense oligonucleotides for 24 hrs. a) Total protein was extracted from a single culture from each treatment group and from the FuGene-only control group. ADAM17 was immunoprecipitated from these extracts and subjected to Western analysis (A = antisense oligonucleotides; Sc = scrambled oligonucleotides; 10 μM). The percentage of ADAM17 in experimental cultures compared to a FuGene-only exposed culture (Fugene) was determined by densitometry as indicated (left panel). The right panel was overexposed to verify the location of the two, expected ADAM17 bands. Both blots reveal decreased expression of ADAM17 in the two cultures exposed to antisense oligonucleotides. b) Cell number was determined as a measure of proliferation (n = 6, *p < 0.05 compared to appropriate control, †p < 0.01 compared to appropriate IL-13-treated, scrambled oligo sample), and c) the amount of TGFα in the supernatant was quantified via ELISA (n = 4, *p < 0.05 compared to appropriate control, †p < 0.01 compared to appropriate treated, scrambled oligo sample).