FIGURE 4.

Confocal imaging of CHERP in live cells. A, colocalization of CHERP(1–916)-GFP with DAPI counterstaining (0.1 μg/ml, 1 min) in HEK293 cells (top) and SKBR3 cells (bottom). B, targeting of CHERP(1–916)-GFP to nuclear subdomains in HEK293 and SKBR3 cells. Subnuclear densities are highlighted (arrows). C, staining of endogenous CHERP in HEK293 cells at low (left) and high magnification (right). Cells were co-stained with SC-35 and DAPI as indicated. D, a detergent-resistant fraction of CHERP in subnuclear foci. Left, immunofluorescence image of HeLa cells transiently transfected with a construct encoding Myc-CHERP and pre-extracted with 0.2% Triton prior to fixation. Right, immunofluorescence image overlaid with a phase-contrast image of the same cells. E, immunofluorescence image of HeLa cells transiently transfected with a construct encoding Myc-CHERP (left), co-stained for endogenous SC35 (middle). Pre-extraction of cells with detergent reveals large CHERP foci. Overlay (right) shows that the majority of, but not all, SC35-foci overlap with CHERP foci (white arrowhead). Boxes circumscribe the region enlarged (3 times) in the lower panel. F, CHERP does not localize to Cajal bodies. Lower (top) and higher (bottom) immunofluorescence images of the distribution of Myc-CHERP (left) and p80-coilin (middle) in HeLa cells. Merged images are shown on the right. G, cells treated with 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) (100 μm for 2 h, bottom) show enlarged CHERP foci that colocalize with SC35, compared with untreated cells (top).