To investigate the roles of Ubc3 in regulation of the HLEC cell cycle, the cells were infected with equivalent levels of viruses which expressed GFP along with nothing, mtUbc3, wtUbc3 (
Fig. 2A , left), and mtUbc2 (
Fig. 2A , right). HLECs infected with empty adenovirus showed indistinguishable patterns and timing of progress through the different phases of the cell cycle compared with the control HLECs (data not shown). The G
1/S transition in the initial cell cycle occurred at 16 hours after release from G
0 and the M/next G
1 transition occurred at ∼38 hours after release from G
0 (data not shown). This is corroborated by the observation that at 40 hours more than 50% of the cells are in G
1 phase, and this proportion increased at 44 hours as more cells move into the G
1 phase of the next cell cycle. In comparison, expression of mtUbc3 resulted in lower proportions of cells in G
1 and a greater proportion of the cells at G
2/M of the first cell cycle (
Fig. 2A , 40 hours). Arrest of the mtUbc3-infected HLEC in G
2/M of the first cell cycle is corroborated by the observation that fewer of these cells progressed to the G
1 phase of the next cell cycle at 44 hours. The G
2/M arrest is curious, since most previous studies indicated roles for Ubc3 in the G
1/S transition, and it might be anticipated that when mtUbc3 is expressed, the greatest proportion of cells would be expected to be in the G
1 phase. The proportion of mtUbc3-expressing cells that are arrested at the G
2/M phase and of the empty-virus–infected cells that progress to the next cell cycle are actually more dramatic, because approximately 25% of the confluence-synchronized cells do not reenter the cell cycle. Of interest, HLECs in which wtUbc3 was expressed also showed increases in the proportion of cells in the G
2/M phase, along with proportional decreases in the fraction of cells at the G
1 phase. These data are due to specific effects of Ubc3, as demonstrated in
Figure 2A , right panel, since cells that were infected with the same amount of adenovirus that encodes mtUbc2 did not show a delay at G
2/M. These results were not due to unequal viral loads, because GFP levels of all three samples are indistinguishable
(Fig. 2B) .