Figure 3

TSPY accelerates G₂/M transition in cell cycle. Flow cytometry analysis of HeLa Tet-off cells harboring either the vector, pTIG, (A) or pTIG-TSPY (B) construct showed a reduced number of cells at G₂/M phase in cells over-expressing TSPY (B, -Dox) than those repressing TSPY (B, +Dox) or harboring the vector alone (A, +/- Dox). To evaluate the probable reason(s) for such differential distribution of cells in various phases of the cell cycle, HeLa Tet-off cells harboring either the vector alone (C) or pTIG-TSPY construct (D) were synchronized at G₁/S, released into S phase and analyzed similarly with flow cytometry at 0, 6, 12, 24, 36 and 48 hours after the release. HeLa cells harboring pTIG-TSPY construct (D) progressed at a faster rate than those harboring the vector alone (C), and reached in the second S phase within 24 hours after release from the synchronization while significant number of cells harboring the vector alone (C) remained in G₁ phase at the same time. Such synchronized cell cycle progression disappeared towards 36 and 48 hours in both cell populations (C, D). When the cells were synchronized at the G₂/M phase, released into mitosis, and analyzed similarly with flow cytometry, such accelerated rate of cell cycle progression were not observed readily between populations harboring the vector alone (E) and TSPY (F). Again, cells over-expressing TSPY showed a reduced number(s) at G₂/M (e.g. 24 and 36 hour time points in D and all time points in F as compared to those in C and E respectively) when they were over-expressing TSPY, resembling those of asynchronous cells (B).