Fig. 3.
PRC1 nuclear localization in female somatic cell lines. (a–h) Immunofluorescences with the indicated antibodies were performed on U2-OS (a), IMR90 (b), and 293 (c–e) cells. Combined Xist FISH and immunofluorescences of 293 cells (f and g) and mouse embryonic female fibroblasts (h). For a and h, colocalization was analyzed by sequential scanning confocal microscopy; for b–g, epifluorescence microscopy was used. (i) PRC1 localization at the Xi is cell cycle-dependent. Asynchronously growing 293 cells were treated with BrdUrd for 1h. Then, cells were fixed and stained with antibodies against CBX4/HPC2, RNF2/RING1B, or MACROH2A1 and BrdUrd and with DAPI (see Material and Methods). Based on the staining of the incorporated BrdUrd into the nascent DNA, different stages of S phase were established. The patterns of distribution of replication sites are shown in Upper. More than 200 cells were scored for CBX4/HPC2, RNF2/RING1B, or MACROH2A1 at the Xi and classified into these different stages of the S phase. (j) 293HEK cells were synchronized in S phase with thymidine, labeled with BrdUrd for 15 min, and released from the blockade for the indicated times. Cells were then fixed and stained with anti-BrdUrd antibody and propidium iodide. Cell cycle profile and BrdUrd incorporation were analyzed by flow cytometry. In parallel, cells growing on glass slides underwent the same thymidine treatment. Then, cells were fixed and immunostained. More than 350 cells were scored for the presence of BMI1, RNF2/RING1B, or MACROH2A1 bodies at the Xi.