Fig. 5. Cell cycle regulation in immortal cells that lack Cdkn2a and Tsg101.

A, the tumorigenic, Tsg101-anti-sense-expressing SL6 cell line and its parental mouse 3T3 fibroblast cell line lack expression of p19^Arf and p16^Ink4a. Cdkn2a-deficient cells were used as negative controls, and immortal p53^−/− MEFs served as positive controls for p19^Arf and p16^Ink4a protein expression. B, Western blot analysis of Tsg101 and Cdkn2a to monitor effective Cre-mediated down-regulation of Tsg101 in MEFs lacking one or two copies of the Cdkn2a gene and their controls. The steady-state levels of Mdm2 did not decrease and were slightly elevated in cells lacking Tsg101. C, flow cytometric analysis of the DNA content in cells doubly deficient in Tsg101 and p19^Arf (Tsg101^fl/fl Cdkn2a^−/− pBabe-Cre) and their controls. The sub-G₁ population of apoptotic cells was gated out in this assay. P19^Arf-deficient cells deprived of essential growth factors (Tsg101^fl/fl Cdkn2a^−/− 0.1% fetal bovine serum (FBS)) served as an additional positive control to monitor the accumulation of cells at G₁ and the relative decrease in the number of cells at the S-phase of the cell cycle. Note that the deletion of Tsg101 in a p19^Arf-deficient background does not cause a cell cycle arrest, whereas growth factor withdrawal from these cells results in a sharp reduction of cells in S phase.