PB numbers increase as cells reach S/G2 phase of the cell cycle.

(A) The Fucci markers mCherry-Cdt1 (red) and AmCyan1-Geminin (cyan) were expressed in U2OS cells and then cells were stained with an anti-Hedls (green) antibody to mark PBs. (Bar 20 µm). It was possible to detect the cell cycle phase using the intensity combination of the red and cyan markers in the cell, as explained in scheme below. (B) The number of PBs in each cell was counted and assigned a cell cycle phase according to the Fucci colors. The plot designates the average PB number in each phase (G1 n = 40, G1/S n = 15, S/G2 n = 40, M n = 10). Error bars represent STDEV and a T-Test was performed. (C) U2OS cells stably expressing GFP-Dcp1a were co-transfected with AmCyan1-Geminin and mCherry-Cdt1 and imaged for 15 hours. Frames show the cytoplasmic GFP-Dcp1a signal together with nuclear AmCyan1-Geminin staining that looks green due to the filter used. The plot represents the relative intensity analysis of all markers as quantified throughout the movie. Red – mean Cdt1 intensity, cyan – mean Geminin intensity, green – number of PBs.