A-H) Tet-off regulation of TSPY and EGFP expression in HeLa cells. A bicistronic transgene capable of expressing both TSPY and EGFP in the same transcriptional unit was stably transfected to HeLa Tet-off cells. EGFP fluorescence (A) and TSPY (B) were detected by direct observation and immunofluorescence respectively. Such expression (i.e. EGFP in C) could be suppressed by administration of doxycycline in the culture media (D). EGFP was located in both cytoplasm and nuclei of the host cells (E, G) while TSPY could display scattered locations along the periphery and within the nuclei, in addition to its cytoplasmic distribution (F, H, blue arrows). I-L) Effects of TSPY expression in cell transfection efficiency and proliferation in HeLa and NIH3T3 Tet-off cells. HeLa (I) and NIH3T3 (J) Tet-off cells were co-transfected with either TIG-TSPY (TSPY) construct or the vector pTIG alone with the TK-Hyg plasmid and were selected in culture media with hygromycin with (+) or without (-) doxycyclinc (Dox). Both HeLa (I) and NIH3T3 (J) cells expressing TSPY (i.e. TSPY without doxycycline) consistently formed higher numbers of colonies than those repressing the same transgene (i.e. TSPY with doxycycline) while those expressing EGFP in the vector (pTIG) alone did not show any differences in the transfection efficiency in the absence or presence of doxycycline. Cell proliferation assays demonstrated that HeLa (K) and NIH3T3 (L) Tet-off cells over-expressing TSPY (without doxycycline) proliferated at a faster rate(s) than those repressing the TSPY gene (with doxycycline). Again, cells harboring the vector (pTIG) alone did not show any difference in their proliferative activities in media with or without doxycycline. Bars in D and H represent 40 μM in A-D and 10 μM in E-H respectively. * Student's t-test analysis indicated that there were statistical significance in differences between TSPY-expressing cells and vector-alone cells.