Cyclin D1 and phospho-cyclin D1 staining patterns. (A) MRC5 cells were pulsed with BrdU and stained as described in Figure 1. Fluorescence photographs of BrdU, cyclin D1 and DNA stains for a single group of cells are presented. Separate cells stain for cyclin D1 and BrdU. (B) NIH3T3 cells were fixed and stained with antibodies against total cyclin D1, or cyclin D1 phosphorylated on Thr-286. This procedure was performed on untreated cells, or following a three hr treatment with MG132 to block proteasomal degradation. Fluorescence images of the same area of cells are presented, along with DAPI stained DNA. No accumulation of cytoplasmic cyclin D1 or phospho-cyclin D1 is apparent in any of these cells.