Figure 1.

PTEN protects stalled replication forks and suppresses genomic instability. (A) Experimental design of the DNA fiber assay. Lengths of nascent replication tracts labeled with IdU were measured by DNA spreading after 5 h of replication stalling with or without HU treatment. IdU tracts in Pten^−/− MEFs show significant strand shortening with HU treatment as compared with Pten^+/+ MEFs. IdU tract lengths reflect replication fork stability. (B) IdU tract lengths in PTEN^+/+ and PTEN^−/− HCT116 cells with or without HU treatment. (C, D) IdU tract lengths in PTEN^+/+ and PTEN^−/− HCT116 cells, as well as in Pten^+/+ and Pten^−/− MEFs with different exposure times to HU treatment. (E) Measurement of IdU tracts in PTEN^−/− HCT116 cells untransfected or transfected with Flag-HA-PTEN^WT, PTEN mutants with or without HU treatment. PTEN^WT and phosphatase activity dead mutants of PTEN^C124S and PTEN^G129E were tested for the ability to rescue stalled DNA replication forks caused by PTEN deletion. (F) CldU tract lengths in PTEN^+/+ and PTEN^−/− HCT116 cells upon HU treatment. CldU tracts represent continuous tracts formed during HU treatment. (G) Gap lengths between IdU tracts prior to HU treatment and CldU tracts after HU treatment in PTEN^+/+ and PTEN^−/− HCT116 cells. Gap lengths indicate the efficiency of blocking the restart. (H) Diagram of gap lengths between IdU tracts before HU treatment and CldU tracts after HU treatment (leading strand) and CldU tract lengths with HU treatment (lagging strand) showing there is asymmetric replication fork movement in PTEN^−/− HCT116 cells. (I) Chromosomal aberrations measured by metaphase chromosome spreads in PTEN^+/+ HCT116 and PTEN^−/− HCT116 cells with or without HU treatment. Representative images of chromosomal aberrations in metaphase chromosomes are shown. Cells with PTEN deletion show genomic instability under replication stress. All data are presented as means ± SEM and analyzed by unpaired t-test. ^**P< 0.01^**P< 0.001.