Fig. 3: APC/C facilitates RECQ4 Q757X replication complex formation.

a Flow cytometry analysis of parental HEK293 cells after release from nocodazole blocking at the indicated time points. The cells were labeled with BrdU and co-stained for BrdU incorporation (Y-axis) and DNA content (propidium iodide [PI], X-axis). Percentages of cells in G₁, S, and G₂/M phases are shown. Percentages of cells in G₁, S, and G₂/M phases are shown, with the highest percentage in each time point indicated in red. b Representative western blot analysis of the indicated proteins in whole-cell extracts (WCE; input; left) prepared from cells shown in (a) and of proteins co-immunoprecipitated (right) with RECQ4 using a rabbit anti-RECQ4 antibody at the indicated time points post-nocodazole (Noc) release. c Quantification of fold change in the amount of the indicated co-purified proteins normalized to the bait protein RECQ4 compared to 0 h in (b). A higher value on the Y-axis corresponds to an increasing interaction. d Flow cytometry analysis of RECQ4 knockdown (KD) HEK293 cells stably expressing FLAG-tagged RECQ4 wild type (WT, top) or Q757X mutant (bottom) protein after release from nocodazole blocking at the indicated time points. Percentages of cells in G₁, S, and G₂/M phases are shown, with the highest percentage in each time point indicated in red. e Representative western blot analysis of the indicated proteins in WCE (left) prepared from cells shown in (d) and of proteins co-purified with FLAG-RECQ4 WT or Q757X mutant proteins using M2 agarose beads (right) at the indicated time points post-nocodazole release. * indicates non-specific bands cross-reacting with the GINS4 antibody. f Representative western blot analysis of APC5 in FLAG-RECQ4 WT or Q757X mutant expressing cells treated with or without shRNA specifically targeting APC5 [APC5 knockdown (KD)]. Actin was used as a loading control. g Representative western blot analysis of the indicated proteins co-purified with FLAG-RECQ4 WT or Q757X mutant proteins prepared from cells shown in (f). Source data are provided as a Source data file.