Skip to main content
. 2005 Nov;25(21):9350–9359. doi: 10.1128/MCB.25.21.9350-9359.2005

FIG. 3.

FIG. 3.

Fluorescence redistribution after photobleaching analyses of GFP-hPCNA before DNA damage induction. Cells stably expressing PCNA fused to GFP were subjected to a local bleach pulse, and the kinetics of fluorescence recovery in the bleached area was determined. (A) The fluorescence in a small strip spanning the entire nucleus was bleached with a 200-ms high-intensity laser pulse. The recovery of fluorescence in the strip was monitored at intervals of 100 ms, and the measured fluorescence intensities over time were plotted. This photobleaching protocol was applied to GFP-hPCNA-expressing cells in the different stages of the cell cycle. To determine the immobile fractions, the final measured fluorescence intensity was normalized to the prebleaching pulse fluorescence intensity. (B) Diffusion coefficient of GFP-hPCNA in different stages of the cell cycle. To determine the Deff, the fluorescence intensity immediately after bleaching and the final postbleaching pulse fluorescence intensity measured were normalized between zero and one. The Deffs in the different stages of the cell cycle were determined by fitting the experimentally obtained curves to a mathematical model describing diffusion (6). Measurements were performed in triplicate, and consistent results were obtained among different sets of experiments. Error bars indicate twice the standard errors of the mean. (C) Fluorescence loss in photobleaching and fluorescence redistribution after photobleaching of GFP-hPCNA in different stages of the cell cycle. The lower region of a cell containing replication foci was bleached by a single laser pulse. The cell was imaged at the indicated times after bleaching. FLIP was measured in the unbleached half of the cell, while FRAP was measured in the bleached half of the same cell. The same experimental protocol was applied to cells in the early, mid-, and late S and G1/G2 phases of the cell cycle. (D) Quantitation of the simultaneous FRAP and FLIP experiment on GFP-hPCNA replication foci. “n” represents the number of cells examined.