Figure 7. Knockdown of ZSCAN5A increases number of cells entering mitosis and aneuploidy.

HEK-293 cells engineered to stably carry an inducible short hairpin RNA construct targeting ZSCAN5A (ZSCAN5A-Tet-shRNA), or empty-vector control were incubated with 1 μg/mL doxycycline (Dox) for 48h on gelatin-coated glass cover slips. A. Fixed coverslips were stained with mitotic marker anti-Phospho-Histone H3 (Ser10) antibody (EMD Millipore), and DAPI to visualize mitotic cells using microscopy, examining totals of more than > 2000 cells per cell line. The number of mitotic nuclei was significantly increased in the shRNA-carrying cells (p = 5.04e-12, two-sample test for equality or proportion without continuity correction; see Methods), with standard errors from the counting of multiple samples for each cell type shown as error bars. B. Flow cytometry of control and C. ZSCAN5A-Tet-shRNA cells taken 48 h after Dox treatment consistently revealed the appearance of a small population of cells (average of 2.01%) with lower DNA content compared to normal G1 cells after ZSCAN5A knockdown (arrows in C). This population was never detected in the Dox-treated (B) or untreated (not shown) control cells we tested. In both experiments, ZSCAN5A knockdown rate was determined by qRT-PCR to be 60 % (A) or 43% (B) respectively.