Fig. 4

RETSAT recruits DDX39B onto replication forks to resolve R-loop. A Treatment and sample collections used for iPOND combined with LC-MS/MS detection. B Overlapping analysis between absent proteins in RETSAT-KO groups and DNA damage response protein dataset. C Heatmap of DDX39B based on its abundance in LC-MS/MS results. D Co-immunoprecipitation using anti-RETSAT antibody combined with immunoblotting to confirm the interaction of RETSAT and DDX39B in PANC-1 cells under indicated treatments. GAPDH was included as a negative control. E Co-immunostaining of DDX39B (green) and pulse labeled BrdU (red) in parental and RETSAT-KO PANC-1 cells. F Immunoblotting of DDX39B in nuclear and cytoplasmic extractions of PANC-1 cells with or without RETSAT. GAPDH and H2B were used as a cytoplasmic and nuclear loading control respectively. G iPOND combined with immunoblotting to examine DDX39B loading dosage on replication forks in parental and RETSAT-KO PANC-1 cells. H, I Representative images (H) and quantification (I) of R-loop dot blotting using S9.6 antibody in parental and RETSAT-KO PANC-1 cells under indicated treatments. Anti-dsDNA antibody was used as a loading control. J, K Representative images (J) and quantification (K) of R-loop accumulation in parental and RETSAT-KO PANC-1 cells under indicated treatments. L Dot Blotting of R-loops using S9.6 antibody in parental and RETSAT-KO PANC-1 cells with or without RNase H1 ectopic expression. Anti-dsDNA antibody was used as a loading control. M, N Representative images (M) and quantification (N) of γH2A.X positive BrdU foci in RETSAT-KO PANC-1 cells with or without ectopically expression of RNase H1 under indicated cultural conditions. Scale bar = 10 μm. 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