FIG. 2.

Variant population of HeLa cells resistant to Tax-induced senescence. (A) Flow cytometry histograms of nocodazole-treated, Ad-tTa- or Ad-Tax-transduced HeLa cells. For flow cytometry and immunoblots, 1 × 10⁶ HeLa and HeLa/18x21-EGFP cells were grown, respectively, in 15 ml Dulbecco modified Eagle medium (DMEM) plus supplements in a T75 flask. Cells were then treated with adenovirus vectors and nocodazole. Round cells were detached from the flask by vigorous shaking, transferred to a 50-ml tube, and washed twice with 15 ml of phosphate-buffered saline (PBS) solution. Cells that remained attached were trypsinized, collected, and washed twice with PBS. Cell pellets were resuspended in 1 ml PBS, and the cell numbers in each population were counted using a hemacytometer. The majority of Ad-tTa-transduced cells detached from the plate after nocodazole treatment [Ad-tTa(D)]. Approximately one-third of the nocodazole-treated, Ad-Tax-transduced cells were detached from the flask. The Ad-Tax-transduced cells were divided into detached [Ad-Tax (D)] and attached [Ad-Tax(A)] populations. (B) Flow cytometry histograms of asynchronously grown HeLa cells transduced by Ad-tTa and Ad-Tax. For flow cytometry, HeLa cells were fixed in 1% paraformaldehyde, permeabilized with ethanol, treated with DNase- and protease-free RNase A (catalog number EN0531; Fermentas), and stained with propidium iodide (PI) as previously reported (20). Detection of EGFP and PI fluorescence was carried out using a fluorescence-activated cell sorter (Beckman-Coulter Epics XL-MCL flow cytometer). The fractions of cells in G₀/G₁, S, and G₂/M were computed using the ModFit LT software package. (C) Immunoblots of cell lysates prepared from Ad-Tax-transduced HeLa/18x21-EGFP cells that detached from [lane (D)] or remained attached to [lane (A)] the culture dish after nocodazole treatment. The lysate of asynchronously grown cells (lane As) was included for comparison. The antibodies used for the blots are as indicated.