Fig 2: Removal of p53 from promoters independent of proteasome activity. (A–C) To inhibit the proteasome for a brief period, SJSA cells were treated with the pharmacological proteasome inhibitor 20 µM MG-132, or DMSO alone, for 20 min prior to Nutlin washout. Proteasome inhibition was continued after Nutlin was removed. (A) The delayed degradation of p53 as well as the accumulation of ubiquitinated p53 upon proteasome inhibition is visible in representative immunoblots. HIF-1a, a protein with particularly high turnover [22], was detected to control proteasome inhibition. (B) A brief pulse of MG-132 increases the amount of DNA-bound p53 but does not compromise p53-removal from promoters upon Nutlin washout, as revealed by ChIP analysis. Three biological replicates, mean + SEM. (C) The same data as in (B) but normalized as described for Figure 3E, visualizing comparable removal of p53 from its cognate promoters regardless of short proteasome inhibition. *** p < 0.001; ** p < 0.01; * p < 0.05; n.s., non-significant. “-“, no Nutlin washout.

Fig 3: Induction of p53-bound genes by Nutlin and Wip1 inhibitionA. Heat map of p53 at transcription start sites of genes in MCF-7 cells after Nutlin treatment. Chip data on p53-promoter-associations are displayed, red color reflecting the degree of association with p53. Group 1 indicates those genes which were significantly upregulated by both Nutlin alone and Nutlin+Wip1i treatment in the RNA-Seq analysis from Figure 4. Group 2 indicates those genes that were significantly upregulated upon Nutlin+Wip1i treatment but not by Nutlin alone. Group 3 indicates those genes that were significantly downregulated upon Nutlin and Nutlin+Wip1i and group 4 indicates those genes that were downregulated upon Nutlin+Wip1i treatment but not by Nutlin alone. B. Profiler image of p53 occupancy at transcription start sites of genes in MCF-7cells after Nutlin treatment. The profiler image (right) provides the average p53 signal obtained ± 3kb from the transcriptional start site for the genes at each of the above-mentioned conditions. ChIP-seq track data for Nutlin-3a-stimulated MCF-7 cells was obtained from p53 ChIP-Sequencing [40] and downloaded from the Gene Omibus database (ID GSE47043). C. Heat map of p53 on the TSSs of genes dependent on both p53 activity and stability. For better evaluation, we distinguished the genes from A, group 1 and 3, into two classifications, i. e. genes that were induced/downregulated further by the combined treatment in comparison to Nutlin alone (1 and 3), and the ones which were already induced/repressed by Nutlin alone to the maximum extent (2 and 4). D. Profiler image of p53 occupancy at TSSs of genes dependent on p53 activity and stability. The p53 promoter signal was aggregated along the TSSs of these genes as described in C, and a profiler image is displayed on the right.

Fig 4: EGCG inhibits EBV lytic replication by suppressing LMP1-mediated MAPK signal axes in AGS-EBV and B95.8 cells. AGS-EBV and B95.8 cells were starved in 0.1% FBS HAM’s/F-12 or RPMI-1640 medium for 24 h and then treated with different concentrations of EGCG as indicated or vehicle [dimethyl sulfoxide (DMSO)] for 24 h. (A, B) Western blotting analysis was performed to determine LMP1, total and phosphorylated ERKs, p38, and JNKs, total and phosphorylated p53, and total and phosphorylated c-Jun. (C) Effects of EGCG on the expression of Zta and Ea-D. ß-Actin was used as an internal control. (D) Reverse transcription (RT)-qPCR quantification of mRNA expression levels for BZLF1 and BMRF1 genes. (E) Flow cytometry was performed to analyze the effects of EGCG on the numbers of Zta- or Ea-D-positive cells. Incubation with vehicle but no primary antibody followed by incubation with the secondary antibody was used as a negative control. (F) The changes in EBV genome copies were analyzed using qPCR. The levels of EBV DNA copy number from the control group were set to 1. *p < 0.05, **p < 0.01, ***p < 0.001 versus control group.

Fig 5: Depletion of Ajuba results in activation of the DNA damage response. (A) Staining for p53BP1 foci on cells fixed 72 h after transfection with siRNA #3, and siGFP as a negative control. Quantification of the number of p53BP1 foci on three independent siRNA experiments shown in (B). (C, D) Western blots for induction of Chk1 phosphorylation and induction of p53 phosphorylation, showed for the three target sites, 72 h after siRNA transfection.