Journal: Nucleic Acids Research
Article Title: Regulation of p21/CIP1/WAF-1 mediated cell-cycle arrest by RNase L and tristetraprolin, and involvement of AU-rich elements
doi: 10.1093/nar/gks545
Figure Lengend Snippet: RNase L-mediated cell-cycle arrest and p21 induction. ( A ) Flow cytometric analysis of RNase L polyclonal and control cells. Cells were synchronized at the G 1 phase by confluency and aphidicholin treatment for 18 h. Cells were released by 15% FBS for 6 h and were then subjected for flow cytometry. ( B ) Flow cytometric analysis for cell-cycle profiles of RNase L +/ + and RNase L −/− mouse embryonic fibroblasts. ( C ) Expression of p21 and p53 proteins in RNase L-expressing HeLa cells and vector-expressing cells. Cell lysates were subjected to western blotting using specific antibodies. The p21 band intensities were quantified using (ImageJ 1.44 software), and β-actin normalized fold differences were calculated ( right panel ). The results are represented as the mean ± SEM of two independent experiments. ( D ) RNase L + / + and RNase L −/− MEF cells lysates were subjected to western blotting for p53 and p21 levels, using specific antibodies for p53 and p21. The results of β-actin normalized band intensities are represented as the mean ± SEM of two independent experiments. ( E ) Wild-type and RNase L-deficient cells were treated with 10 Gy of γ-radiation and incubated for the indicated times. Total cell lysates were analyzed for western blotting, using specific antibodies for p21 and p53.
Article Snippet: Confluent cells maintained for a period of three days were synchronized at G 1 /S by treating cells with 5 µg/ml aphidicholin (Sigma) for 20 h. Then, cells were washed with phosphate-buffered-saline (PBS) and released into the S phase in complete medium with 15% serum.
Techniques: Flow Cytometry, Expressing, Plasmid Preparation, Western Blot, Software, Incubation
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