Bcl-3 knockdown cells are multinucleated. A) Nuclei of HeLa cells expressing a control shRNA (left panels) or Bcl-3 shRNA (right panels) as described in Fig. 1 were stained with ethidium bromide after five population doublings. Arrow shows a multinuclear cell. B) Box-and-whisker plots of nuclear area from a thousand nuclei of HeLa cells expressing a control shRNA or Bcl-3 shRNA as described in Fig. 1. Nuclei were stained with ethidium bromide. These results were obtained in the described cells after five population doublings. The plots show quartiles, sample minimum and maximum. *p < 0.01 by Wilcoxon rank test. C) Table showing the percentage and standard deviation of mono, bi or multinuclear cells derived from two hundred cells counted in three different preparations of the described cells after five population doublings. D) HeLa cells were transiently transfected with a plasmid encoding a shRNA directed toward Bcl-3 and, along, the ZsGreen protein marker (plasmid pSIREN-RetroQ-ZsGreen, Clontech; CA, USA) (Bcl3). As control, a plasmid with encodes a scrambled shRNA and the same marker, was transfected (Ctrl). After 5, 12 or 18 population doublings (PD), green multinuclear cells were counted (percentage at the bottom) n.d.: not detected. RNA was also isolated from these passages and assayed for Bcl-3 expression by RT-PCR. Note that at the 18th PD, the Bcl-3 knockdown effect was lost. Transfection efficiency was approximately 30%. The differences observed between the experiments in Fig. 1 and 2 were probably due to the lower efficiency of the knockdown strategy used (infection versus transfection).