Figure 2.

Effect of TAT-NPM fusions on cell proliferation and senescence. (A) TAT-NPMΔC fails to suppress senescence in MEFs. WT or Fancc−/− MEFs at passage 4 were incubated with BSA, TAT-NPM, TAT-NPMΔC, or TAT-NPMΔN (30 μg/mL each) for 6 days, with medium and the proteins changed each day. Cells were then stained for SA-β-gal. The graphs on the right are percentages of the cells stained positive for SA-β-gal quantified by counting a total of 100 cells in random fields on a slide. The data represent the mean plus or minus SD of 3 independent experiments. *Statistical significance between TAT-NPMΔC and BSA samples at P < .05. (B) TAT-NPMΔC inhibits cell proliferation. Cells were plated in 96-well plates at a density of 2 × 10³ per well for overnight. Cells were incubated with the indicated proteins (30 μg/mL) and were changed each day. Cell proliferation was determined at the indicated time points. Data represent mean plus or minus SD of 3 experiments. *Statistical significance between TAT-NPM and BSA or between TAT-NPMΔC and BSA samples at P < .05. (C) Cells described in panel A were analyzed for apoptosis, as determined by flow cytometry for the percentage of cells with active caspase 3 (gated in R3). Shown are the representative data of 3 independent experiments with similar results. (D) Uptake of TAT-NPM and TAT-NPMΔC in human cells. HEK293 cells were incubated with 30 μg/mL of the indicated proteins for 30 minutes. After fixation, cells were counterstained with DAPI and the cellular distribution of the FITC-labeled TAT-fusions was visualized with fluorescence microscope. (E) Time course of TAT-NPM protein uptake by human cells. HEK293 cells were incubated with TAT-NPM or TAT-NPMΔC (30 μg/mL each) at the indicated time. Relative FITC intensity was determined by normalizing fluorescence intensity of each treatment with cell numbers. (F) Subcellular localization of the TAT fusions in human cells. HEK293 cells were incubated with TAT-NPM or TAT-NPMΔC (30 μg/mL each) for 30 minutes, washed extensively, and incubated in the absence of the TAT fusions for 60 minutes before fixation. The cells were then stained with anti-His₆ antibody and visualized with fluorescence microscope.