Figure 1: PTEN depletion induces FANCD2-associated anaphase bridges and premature S phase exit.

(a) Anaphase errors increase in PTEN-depleted cells. HeLa cells with and without PTEN shRNA (shPTEN and shControl, n=128 and 103, respectively) were examined by immunofluorescence for mitotic defects. Percentages of mitotic cells with anaphase bridges and micronuclei (arrowheads) are summarized in the histogram with represent 4,6-diamidino-2-phenylindole (DAPI) images shown on the left. Data are presented as means±s.e.m. and analysed by unpaired t-test. *P<0.05. (b) Specification of pre-mitotic errors originating from DNA replication barrier. Immunofluorescence of FANCD2 was employed for labelling damaged DNA in mitosis reflective of replication defects. CENPA and DAPI were used to indicate centromeres and chromosomes. Top panel; anaphase and telophase cells without prominent FANCD2 foci. Mid and bottom panels; cells in anaphase or telophase with pronounced FANCD2 foci. Brackets, paired foci; Arrowheads, single or accumulated foci; Scale bar, 5 μm. (c) Frequencies of FANCD2-associated ana-telophase bridges summarized from FANCD2 immunofluorescence in cells with or without shPTEN in the presence and absence of aphidicolin (APH) treatment (0.1 μM, 24 h). Data are presented as means±s.e.m. (n>50 for each column) and analysed by analysis of variance (ANOVA) with Turkey’s multiple comparisons test. *P<0.05; **P<0.01. (d) APH-treated HeLa cells with and without shPTEN were immunostained as in b. Mitotic cells were analysed for FANCD2 foci numbers and shown in a scatter plot. Data were processed by ANOVA and statistical significance was determined by Turkey’s multiple comparisons test. *P<0.05; ***P<0.001. (e) The distribution frequency of FANCD2 foci number in HeLa cells with and without shPTEN in the absence of APH treatment. (f) PTEN^+/+ and PTEN^−/− DLD-1 cells were treated with 1 μM APH for 0, 2 or 6 h, before 5-bromo-2'-deoxyuridine (BrdU) labelling and propidium iodide (PI) staining for flow cytometry analysis of cell cycle distribution. S phase was divided into three sub-S phases (early-S1, mid-S2 and late-S3) as indicated. (g,h) Summaries of cell cycle distribution in different phases (G0/G1, S and G2/M, g) or sub-S phases (S1+S2 and S3, h), respectively.