Figure 4.

PR-Set7 regulates replication fork activity. (A) Distribution of fork velocity from 200 ongoing replication forks in shLuc or shPR7 U2OS cells and in shPR7 U2OS_SR cells (left). Squares represent the quartiles (25% of the fork rate values smaller and higher than the median value), and the deviations show the smallest and greatest fork rate values. Mean fork rates with SDs are indicated for each sample (right). P < 0.0002 as determined by the nonparametric test of Mann-Whitney. (B) Distribution of replicated track lengths after 30 min of BrdU incorporation in shLuc U2OS (top), shPR7 U2OS and U2OS_SR cells (middle), and shPR7 U2OS treated with caffeine for 5 h (bottom). Mean lengths with SDs are indicated for each panel. Results were averaged from five independent experiments. (C) Quantification of active replication sites for 10 Mb DNA in BrdU-positive shLuc (bar 1) or shPR7 (bar 2) U2OS cells, BrdU-positive shPR7 U2OS_SR cells (bar 3), and BrdU-positive shPR7 U2OS cells treated with caffeine for 5 h (bar 4). The number of active replication sites was obtained by counting the number of individual BrdU tracks for 10 Mb DNA 3 d after shRNA expression in five independent DNA combing experiments. Error bars represent SD. (D) Distances between replicated tracks labeled with BrdU in shLuc and shPR7 U2OS cells, shPR7 U2OS_SR cells, and shPR7 U2OS cells treated with caffeine for 5 h. The mean distances with SDs are indicated (right). (B and D) P < 0.0001 as determined by the nonparametric test of Mann-Whitney. (E) Cell cycle profiles of shPR7 U2OS cells untreated or treated with caffeine 3 d after shRNA expression. At the starting time point (top), cells were untreated (middle) or treated (bottom) with caffeine, and, 5 h later, they were labeled with BrdU before harvest and stained with an anti-BrdU antibody and 7AAD. Only histograms of DNA fluorescence are shown.