FIGURE 2. Mdm4 and Mdm2 act through partially distinct mechanisms.

(A) H1299 cells were transfected with siRNA for 48 h to knockdown Mdm4, Mdm2, or both, and labeled with IdU and CldU as indicated to detect single replication forks.

(B) Representative images of tracks of newly synthesized DNA were visualized by immunofluorescence, staining CldU (red) and IdU (green).

(C) Analysis of IdU-labeled tracks upon depletion of Mdm4 or Mdm2 or Mdm4 and Mdm2 with 10–90 percentile whiskers. Two biological replicates are shown in Suppl. Fig. 2 A–B.

(D) MEFs with four different knockouts (p53−/− , p53−/− Mdm2−/− , p53−/− Mdm4−/− , p53−/− Mdm2−/− Mdm4−/−) were labeled with CldU for 20 min and IdU for 60 min.

(E) Immunostained tracks of CldU (red) and IdU (green) in representative images.

(F) Fork progression in MEFs displayed as a boxplot. A biological replicate is shown in Suppl. Fig. 2 D.

(G) H1299 cells were first transfected with siRNAs to deplete Mdm2 or Mdm4, followed by plasmid transfection to overexpress Mdm2 or Mdm4 after 24 h. After another 30 h, samples were subjected to fiber assay labeling with 25 μM CldU (20 min) and 25 μM IdU (60 min).

(H) Labeled tracks of CldU and IdU stained in red and green, respectively.

(I) Fluorescently labeled tracks displayed as boxplots with 10–90 percentile whiskers. A biological replicate is shown in Suppl. Fig. 2 E.

(J) Labeled tracks were immunostained in red (CldU) and green (IdU).

(K) The fork rate of replication during the IdU label is shown in boxplots. A biological replicate is shown in Suppl. Fig. 2 G.

(L) Mdm4 was ectopically expressed in MEFs for 30 h before fiber assays were conducted.

(M) Representative images of replication tracks from MEFs with single p53 knockouts, p53/Mdm4 and p53/Mdm2 double knockouts as well as triple knockouts, transfected with an expression plasmid for wildtype Mdm4.

.(N) IdU track lengths representing fork progression in MEFs transfected with Mdm4 as in M, displayed in boxplots with 10–90 percentile whiskers. A biological replicate is shown in Suppl. Fig. 2 J.

(O) Fluorescently labeled tracks in red (CldU) and green (IdU).

(P) Analysis of fork progression in the IdU label after Mdm4 depletion and ectopic expression of wildtype Mdm2 or Mdm2 with the mutation C462A, lacking its E3 ubiquitin ligase function. A biological replicate is shown in Suppl. Fig. 2 K.