Figure 3: PTEN is required for restart of DNA replication at stalled forks.

(a) A schematic protocol of dual labelling DNA fibre assay for evaluation of replication restart following HU- or APH-induced fork stalling. (b) PTEN^+/+ and PTEN^−/− DLD-1 cells were pre-labelled with CldU and then treated with 2 mM HU for 2 h, before post-labelling with IdU for monitoring replication recovery. Selected areas are magnified for visual enhancement of stalled CldU tracts and gaps between CldU and IdU tracts in PTEN-null cells. Scale bar, 10 μm. (c,d) Summary of stalled fork frequencies (n>300) and gap lengths (n>200) between stalled sites and their corresponding re-initiation sites. Data are presented as means±s.e.m. and analysed by unpaired t-test. **P<0.01; ***P<0.001. (e) Summary of DNA fibre restart assay in PTEN^+/+ (n=284) and PTEN^−/− (n=336) cells following APH (3 μM) treatment. Data were scored and analysed as in d. **P<0.01. (f) Pten^+/+ and Pten^−/− MEFs were pulse labelled with CldU and subsequently treated with HU (2 mM) or APH (3 μM) for 2 h, followed by IdU pulse labelling to monitor replication recovery. A minimum of 200 fibre units was scored for each criterion. Data are presented as means±s.e.m. and analysed by unpaired t-test. *P<0.05. (g) BrdU incorporation analysis in response to APH treatment. HeLa cells with and without shRNA-mediated PTEN knockdown were subjected to 3 μM APH treatment for different time periods as indicated, before BrdU pulse labelling for 15 min. Cells were then fixed for immunofluorescence of BrdU. Scale bar, 25 μm. (h) Immunofluorescence images from g were analysed for BrdU intensity and summarized as mean intensity per cell (n>150). Data are presented as means±s.e.m. in relative arbitrary unit and analysed by unpaired t-test. NS, not significant; ***P<0.001. (i) Pten^+/+ and Pten^−/− MEFs were treated with 3 μM APH for 30 min followed by BrdU pulse labelling. Immunofluorescence intensities of BrdU were scored before and after APH treatment. Data were analysed by analysis of variance and statistical significance was determined by Turkey’s multiple comparison tests. **P<0.01.