Fig. 3
RETSAT promotes fork restarting under replication stress. A Co-immunostaining of RETSAT (green) and BrdU pulse labeled replication foci (red) in PANC-1 cells cultured under normoxia (21% O2) or hypoxia (0.3% O2). B iPOND assay to validate location of RETSAT on replication forks. PCNA was included as a positive control. C Immunoblotting of RETSAT in PANC-1 and BxPC-3 infected with or without RETSAT CRISPR gRNA lentivirus. β actin was used as a loading control. D, E Representative images (D) and quantification (E) of fork restarting in parental or RETSAT-KO PANC-1 cells under indicated treatments. 2.5 mM Hydroxyurea (HU) was used to induce replication stress. At least 200 single forks were calculated in each sample. F, G Representative images (F) and quantification (G) of replication fork damage in PANC-1 parental and RETSAT-KO cells under indicated treatments. Pulse labeled BrdU foci was indicating DNA replication sites. γH2A.X was used to indicate DNA damage. H, I Immunoblotting of γH2A.X in parental and RETSAT-KO PANC-1 (H) and BxPC-3 (I) under indicated treatments. β actin was used as a loading control. J Quantification of neutral comet assay in parental and RETSAT-KO PANC-1 cells under indicated treatments. At least 150 single comets were calculated in each sample. K Immunoblotting of ATR, p-ATR (Ser428), CHK1, p-CHK1 (Ser345) andβ actin in parental and RETSAT-KO PANC-1 cells under indicated treatments. L Flow cytometry based Annexin V apoptosis quantification in parental and RETSAT-KO PANC-1 cells under indicated treatments. 1 μM PF-477736 was used to inhibit CHK1 activity. M, N Representative images (M) and quantification (N) of clone formation assay of parental or RETSAT-KO PANC-1 cells under indicated treatments. Scale bar = 10 μm in (A), 50 μm in upper four panels and 10 μm in lowest panel in (F). n = 3 independent experiments unless otherwise stated. All data are presented as mean ± SEM. P values were calculated using a two-tailed student’s t test