Figure 4
G1 progression of the cell cycle is markedly hampered in CSN5-depleted cells.
CSN5-floxed p53-null MEFs containing CRE-ER (as in Fig. 3) were cultured with and without 4OHT and passaged in a normal medium for 4 days. Cells were cultured at high density to arrest them in G0/G1 by contact inhibition for 48 hours and replated at low density to reenter the cell cycle. (A) Cells at indicated times were harvested and analyzed for DNA content by flow cytometry. Percentages of cells in S phase are shown. (B) Lysates isolated from cells at indicated times were analyzed by Western blotting using antibodies against CSN5, cyclin D1, cyclin E, cyclin A, CDK2, pRb, Rb phosphorylated at Ser 807 and Ser 780 and γ-tubulin. (C) Total RNA was extracted from cells at indicated times and analyzed by quantitative RT-PCR using a pair of primers specific to cyclin D1, cyclin E and β2 microglobulin (B2M). (D), (E) Cells were cultured with and without 4OHT in the absence and presence of LMB, fixed and stained with an antibody to CDK2 (D) and cyclin E (E). Cells with cytoplasmic signals were enumerated (right panels). DNA was visualized by staining with Hoechst 33342. Original magnification; x200.
