Figure 2

A-B. TNF-α induced arrest of cells in G0/G1 phase and enhanced expression of p21 and p27. A) Monolayers and spheroids were treated with TNF-α (10 ng/ml) for 24, 48, and 72 hr and cell cycle analysis was done by flowcytometry. The percent population of cells in G0/G1, S and G2/M phases of cell cycle was determined using Modfit software. The plot is a representative of three similar experiments. B) Monolayers (M) and spheroids(S) were treated with TNF-α (10 ng/ml) for 3 hr and the expression of p21 and p27 was determined by Western blotting. Blots were reprobed for β-actin and used as control for equal loading of proteins. C. Differential expression and localization of p21 and p27 in LN-18 cells stimulated with TNF-α. LN-18 cells were grown on coverslips for 24 hr and treated with TNF-α (10 ng/ml) for different time periods. Cells were stained with antibody recognizing nuclear and cytoplasmic p21, or with p27 antibody followed by secondary antibody labeled with Cy3. The expression of p21/p27 is depicted as red fluorescence. DAPI was used for nuclear staining (blue). The merged images show nuclear localization of p21 and p27 localized predominantly in the cytoplasm. (Magnification 63×).