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. Author manuscript; available in PMC: 2021 Jun 1.
Published in final edited form as: Oncogene. 2020 May 19;39(25):4828–4843. doi: 10.1038/s41388-020-1325-1

FIGURE 3. Full-length Mdm4 is required for supporting DNA replication.

FIGURE 3.

(A) Schematic diagram of five plasmid constructs expressing either full-length Mdm4, Mdm4 carrying a deletion of its p53-binding and RING domains, or the p53-binding and RING domain alone.

(B) H1299 cells were depleted of endogenous Mdm4 by siRNA transfection for 48 h, transfected with plasmid DNA to overexpress Mdm4 or fragments of it for 30 h, and labeled with CldU (20 min) and IdU (60 min) for fiber analysis.

(C) Replication fork progression upon overexpression of Mdm4 fragments while depleting endogenous Mdm4. Immunostained tracks of CldU (red) and IdU (green) in representative images.

(D) Boxplot analysis of IdU-labeled tracks upon Mdm4 depletion and overexpression of wt-Mdm4 as well as Mdm4 (aa 201–489) or the p53 binding domain alone (aa 1–198) with 10–90 percentile whiskers. A biological replicate is shown in Suppl. Fig. 3 B.

(E) Replication tracks after immunostaining of CldU (red) and IdU (green).

(F) Boxplot analysis of IdU-labeled tracks upon Mdm4 depletion and overexpression of wt-Mdm4 and Mdm4 (aa 1–344) with 10–90 percentile whiskers. Two biological replicates are shown in Suppl. Fig. 3 D–E.

(G) Replication tracks.

(H) Analysis of fork progression upon Mdm4 depletion and overexpression of wt-Mdm4 or the Mdm4 RING (aa 345–489) domain alone. Two biological replicates are shown in Suppl. Fig. 3 G–H.