Glutamine Deprivation Induces Abortive S-Phase Rescued by Deoxyribonucleotides in K-Ras Transformed Fibroblasts (original) (raw)
Figure 3
Expression and localization of p27kip1 in normal and transformed cells.
For p27kip1 expression analysis normal cells (A) and transformed cells (B) grown in media containing 4 mM glutamine, 1 mM glutamine and 0.5 mM glutamine were collected at appropriate time points and 40 µg of proteins from the total cellular extract were subjected to SDS-PAGE followed by Western blotting with p27kip1 specific antibody. One of at least three independent experiments is shown. Relative quantitative analysis of p27kip1 protein levels after Western blot film acquisition by scanner and densitometry analysis using Image J program, as described in Figure 2, in normal cells (C) and transformed cells (D). p27kip1 cellular localization in normal (E, F, G and H), and transformed (I, L M and N) cell lines was analyzed by immunofluorescence staining for p27kip1 (red staining), after 24 and 96 hours of growth in media supplemented with either 4 mM glutamine (left pictures) or 0.5 mM glutamine (right pictures). Nuclei were visualized by DAPI staining (Blue staining). The pictures are the result of a merge between the two single images acquired. At least 200 cells were scored for each sample. The averages of three independent experiments are presented in G, H, M and N with errors bars indicating SD. Bars, 20 µm.