Click on image to view larger version.
Figure 1. Overexpression of cFLIP and Bcl-xL suppressed NF-
B activity in myoblasts. A) Analysis of NF-B binding activity to a consensus NF-B oligonucleotide. Myoblast cells were cultured for 60 min in the presence of 20 ng/ml IL-1ß or for 24 h in the presence of 293 or PC-3 media (1:2 dilution). Nuclear extracts were prepared and assessed for DNA binding activity to a consensus NF-B oligonucleotide by EMSA. Lane markers indicate cells with stable expression of IKBSR, cFLIP, or Bcl-xL, GFP from a GFP expression plasmid or untransduced (Ctl). B) Analysis of NF-B transcriptional activity. Myoblast cells with stable expression of IKBSR or cFLIP (control cells are untransduced) were cotransfected with pNF-B-hrGFP (200 ng/well) and pCMV-ß (100 ng/well). 6 h after transfection, cells were cultured in the presence or absence of conditioned media (1:2 dilution) (left panel) or cytokines [no cytokine (no cyt) or 20 ng/ml TNF-
, IFN-
, IL-1ß, or IL-6] alone (right panel). GFP-positive and ß-galactosidase-positive cells were counted 24 h after treatment. At least 8 randomly selected fields/well were counted (100x). NF-B activity is presented as a ratio of GFP-positive to ß-galactosidase-positive cells expressed as mean ± SEM vs. control myoblast cells. One of at least 4 independent experiments is shown (n=8, P<0.05). C) Analysis of NF-B transcriptional activity in myoblast cells with stable expression of Bcl-xL cotransfected with pNF-B-hrGFP (200 ng/well) and pCMV-ß (100 ng/well). 6 h after transfection cells were cultured in the presence of 293 or PC-3 cell conditioned media (1:2 dilution). GFP-positive and ß-galactosidase-positive cells were counted 24 h after treatment. At least 8 randomly selected fields/well were counted (100x). NF-B activity is presented as a ratio of GFP-positive to ß-galactosidase-positive cells expressed as mean ± SEM vs. control myoblast cells. One of at least 4 independent experiments is shown (n=8, P<0.05).
