Fig. 4

LSF knockdown in HeLa cells results in mitotic defects. a Effects of LSF-specific siRNA on synchronized HeLa cells expressing YFP-labeled H2B were analyzed utilizing time-lapse microscopy. Schematic of experimental protocol for panels b-e. b Representative images of cells treated with 20 nM of either control siRNA (top) or LSF siRNA (bottom). Numbers represent the time (in minutes) for one particular cell in the image from nuclear envelope breakdown (designated as time = 0 for that cell). c Quantitation of mitotic time from nuclear envelope breakdown (NEB) to anaphase for a population of cells treated with control siRNA or siRNA targeting LSF. Mitotic times (mean time in minutes +/− standard error, n) for 20 nM control siRNA, and 5, 10, or 20 nM LSF siRNA were: 57.9 +/− 2.8, 101; 296 +/− 16, 77; 324 +/− 25, 48; and 235 +/− 16, 84; respectively. Standard errors of the mean are based on the number of cells analyzed in a single experiment. d Quantitation of cellular events at increasing concentrations of LSF siRNA during the time lapse microscopy, including the percentage of cells that visually rounded up as expected for mitotic entry (by phase contrast), but were delayed with condensed but unaligned chromosomes, and the percentage that exited mitosis, but with multinucleation “mitotic slippage”. The control had neither of these phenotypes among the cells counted (~ 100 per group). e Bottom: γ-H2AX staining of HeLa cells treated with 20 nM control or LSF siRNA. Top: Representative image of UV-treated HeLa cells as a positive control. All images were captured at the same intensity. Scale bars: 20 μm