Figure 7
Cytoplasmic cyclin E is responsible for induction of senescence.
(A) Wild-type MEFs were cultured in vitro for more than 10 passages. Cells were fixed and stained with antibodies to mouse cyclin E and CDK2. (B) NIH3T3 and HEK293T cells were mock-transfected (None) or transfected with an empty vector (Vector) and a mammalian expression vector containing human cyclin E fused with ER (hCycE-ER). Transfectants were sorted for the GFP-positive signal 4 days after transfection. Lysates isolated from transfected cells were analyzed by Western blotting using antibodies specifically recognizing human cyclin E and γ-tubulin. (C) NIH3T3 cells transfected with hCycE-ER together with GFP were fixed and stained with antibody specific to human cyclin E. Chromosomal DNA was visualized by staining with Hoechst 33342. (D) NIH3T3 cells transfected and prepared as in B were plated (2.5 × 104 cells per plate) and counted after 3 and 5 days. (E) NIH3T3 cells transfected and prepared as in B were plated and stained for SA-β-Gal activity. As a control, NIH3T3 cells arrested in G0/G1 by serum starvation for 48 hours were assayed (-FBS) likewise. (F) HEK293T cells transfected and prepared as in B were plated (2.5 × 104 cells per plate) and counted after 3 and 5 days. (G) HEK293T cells transfected and prepared as in B were plated and stained for SA-β-Gal activity. (H) Transfectants were stained with Hoechst 33342 and observed under a fluorescence microscope. SAHF-like-structure-positive cells were enumerated and percentages are shown. Original magnification; x200 for A and C and x50 for (E) and (G).
