Fig. 1. F9 cell differentiation is accompanied by a specific change in the subnuclear distribution of TIF1ß. (A) Phase-contrast photomicrographs of F9 EC cultures showing undifferentiated F9 stem cells (control) and differentiated endoderm-like cells after 4 day exposure to 1 µM retinoic acid (RA). (B) Confocal images of single optical sections through the nucleus of vehicle-treated (control) and RA-treated F9 cells. The left panels show the Hoechst DNA staining, which highlights A/T-rich repeat sequences present in the centromeric heterochromatin (bright blue patches), and the right panels correspond to immunodetection with specific monoclonal antibodies (Abs), as indicated. Identical results were obtained using two different TIF1ß-specific Abs (PF64 and 1Tb3) raised against distinct epitopes. Bars, 5 µm. (C) TIF1ß colocalizes with all HP1-containing foci in the nuclei of RA-treated F9 cells. Cells were double-labeled with Abs against HP1 (green) and TIF1ß (red). (D) Time course of TIF1ß heterochromatin association following RA addition. F9 cells were plated at a density of 10² to 10³ cells/cm² in the presence of 1 µM RA and were harvested at the time points indicated. Each coverslip was stained for TIF1ß, and cells in 10 to 20 randomly selected confocal fields were scored for the presence or absence of TIF1ß nuclear foci. At least 1000 nuclei were scored per time point. Bars indicate the percentage of nuclei scoring positive for the focal TIF1ß staining pattern characteristic of heterochromatin association, plotted versus time after RA addition. The average results from four independent experiments are depicted.