This system was chosen because it allows dual color imaging, in which G1 phase nuclei are labeled Gemcitabine HCl orange and S G2 M phase nuclei are labeled green. A fluorescent Tax vector was constructed that allows the identification of Tax expressing HeLa Fucci2 cells. This vector con tained Tax, an internal ribosomal entry site, cyan fluorescent protein, and a Flag sequence at the 3 end of tax. The vector was expressed in HeLa cells, and Tax expressing cells were stained with an anti Flag MAb followed by an Alexa Fluor 594 secondary antibody. As shown in Figure 3A, all Tax expressing cells were CFP positive. HeLa Fucci2 cells were plated on a glass coverslip, transiently transfected with Tax IRES CFP or the CFP control vector, and then incubated for 24 h.

Next, fields containing orange, green, and blue fluorescence were selected and images were acquired using an Olympus LCV110 Imaging System. The prolif eration of control HeLa Fucci2 cells was evidenced by the fraction of cells at G1 phase with orange nuclei, the fraction of cells at S G2 M phase with green nuclei, and the subsequent change in the fluorescence of these cells, which indicated that the cells progressed normally through the cell cycle. At 24 h post transfection, all HeLa Fucci2 cells expressing Tax IRES CFP, which resulted in blue fluorescence, also had orange nuclei, indicating that they were in G1 phase. During the culture period, HeLa Fucci2 cells expressing Tax IRES CFP did not progress to S G2 M phase, as evidenced by the presence of orange nuclei and the absence of green nu clei in Tax expressing cells.

Additionally, a marked decrease was observed in the proportion of Tax IRES CFP expressing cells in S G2 M phase com pared with control cells expressing CFP alone, indicating that Tax arrests cells at the G1 phase of the cell cycle. Interestingly, overexpression of Tax appeared to re duce the number of HeLa Fucci2 cells in culture. Moreover, apoptosis was assessed by the ap pearance of rounded cells after an increase in the num ber of Tax expressing cells at G1 phase, starting at 36 h post transfection. At 72 h post trans fection, there was a notable reduction in the overall number of cells, as well as in the percentage of Tax expressing cells.

Expression kinetics of genes involved in cell cycle regulation and apoptosis that are altered following induction of tax protein To analyze the correlation between the expression of genes related to cell cycle regulation and apoptosis with the dynamics of cell cycle and apoptosis, total RNA was prepared at 12, 24, 36 and 48 h after transfection of HeLa cells with Tax or a control vector. Each Drug_discovery RNA sample was then subjected to qRT PCR. As indicated in Figure 4, the expression levels of SMAD3, GADD45A and GADD45B in Tax transfected cells began to increase from 6 h post transfection and reached a peak at 24 h, decreasing again by 36 h.