Figure 6: Regulation of DNA replication is conserved and FAF1-dependent in human cells.

(a) Western blot analysis of replication checkpoint activation by phosho-specific antibodies in HEK293T cells transfected with indicated siRNAs. (b) Schematic illustration of DNA replication fibre analysis—(a) Indicates ongoing replication (red and green arrows), (b) stalled replication forks (red arrows only), (c) newly fired origins (green arrows only) and (d) terminated/converged forks. Sites of DNA replication initiation (I) or termination (T) are labelled, respectively. (c) Western blot analysis of T24 cell lysates transfected with indicated siRNAs. Efficiency of siRNA-mediated depletion is shown for FAF1 protein levels. (d,e) Representative images of microscopic analysis of DNA replication fibres by molecular combing in T24 cells after indicated siRNA transfection. CldU (first pulse) incorporation is shown in red, incorporated IdU (second pulse) in green. Quantification of replicated DNA tract length after indicated siRNA transfection in T24 cells. Tract length was determined for 100 individual forks per condition and experiment. The experiment was repeated twice. Whisker box plots show mean values and data within the 10–90 percentile. (f–i) Representative images of microscopic analysis of DNA replication fibres by molecular combing in U2OS cells after indicated siRNA transfection. Quantification of replicated DNA tract length, stalled replication forks and newly fired dormant origins after indicated siRNA transfection in U2OS cells. Tract length was determined for 100 forks per condition and experiment. Fork stalling and origin firing was quantified for 400 forks per condition and experiment. The experiment was repeated in three replicates. (j) Western blot analysis of control HA and HA-FAF1 co-IPs. Nuclear lysates were prepared from HEK cells expressing the p97-E587Q variant. (k) HEK cells were depleted for control or FAF1 by siRNA. CDT-1 and PCNA levels were monitored at indicated time points after CHX treatment by western blot analysis. Scale bar, 5 μm. Data show mean values. Error bars represent s.d.. The single asterisk indicates a P value of ≤0.05, the double asterisk ≤0.001 and the triple asterisk indicate P values of ≤0.0001.