Fig. 4

Depletion of Ensa extends S phase without significantly altering the overall spatiotemporal patterns of replication. a HeLa chromobody cells were transfected with SC and Ensa siRNA and imaged by time-lapse microscopy for 72 h and S-phase duration visualised by GFP-nanobody-dependent staining of PCNA, recorded and represented as scatter dot blot (n = 18 for siSC and n = 20 for siEnsa). Two-tailed unpaired Student’s t-tests was performed to determine the statistical relevance ***p < 0.0001. Data pooled from three biological replicates. b Representative images of early, mid and late replication factories from HeLa chromobody control cells used for time-lapse microscopy. Scale bar, 10 μm. c Representative images of mid-replication origins from Supplementary Movies 1–6. Images corresponding to three Z-planes of three scramble and three siEnsa-treated HeLa cells stably expressing a chromobody directed to PCNA. Plans correspond to 4 h, 3 h 20 min and 4 h 15 min time points after DNA replication started in siSC-treated cells 1, 2 and 3 and to 5 h 15 min, 5 h 15 min and 6 h 50 min time points in siEnsa-treated cells 1, 2 and 3 from movies on supplementary data. Scale bar, 10 μm. d HeLa chromobody cells treated as in c were imaged by time-lapse microscopy and the length of the early, mid and late S-phase patterns was recorded and represented as a bar graph