Journal: PLoS Pathogens
Article Title: Novel modulators of p53-signaling encoded by unknown genes of emerging viruses
doi: 10.1371/journal.ppat.1009033
Figure Lengend Snippet: (A) p53 signaling is activated either by (i) etoposide or (ii) nulin-3. Etoposide binds and inhibits topoisomerase II, which results in formation of DSB and activates DDR. ATM senses DSB and autophosphorylates. Because SSB are being introduced during DSB repair, etoposide also activates ATR kinase. Both kinases phosphorylate p53 and HDM2, an E3 ubiquitin ligase, which breaks their interactions and leads to p53 accumulation and stabilization and results in expression of p53 target genes. Nultin-3 inhibits HDM2:p53 interactions and thereby induces p53 accumulation. The response is measured using Luc-reporter assay and immunoblotting with Abs specific for ATM pSer1981, ATR pThr1989, p53 pSer15, p53 and p21. (B) Relative expression levels of p53-dependent luciferase reporter. Shown is the mean effect size plus 95%CI after adjustment for multiple comparisons to control using Dunnett’s methods. The mean effect was calculated by linear model of three independent biological replicate transfections/treatments. For each transfection, three technical replicates were measured. Orfs with confidence intervals not overlapping “0” were considered significant hits (p≤0.05). The names of each tested orf are shown on the left. For detailed description see Experimental Procedures and . KSHV orf10f (3xflag-tagged, pOME0004), KSHV orf10l (lentivirus, pOME0004L), KSHV orf45f (3xflag-tagged, pOME0016), KSHV orf45l (lentivirus, pOME0016L), MERS-CoV orf8b (pOME0215), ZIKV NS2A (pOME0303R), ZIKV NS2Af (3xflag-tagged, OME0304), ZIKV NS2Al (lentivirus, pOME0304L). For KSHV orf25g and KSHV orf45g see Experimental Procedures. (C) Cell viability for EV (pCMV-Neo-Bam), p53-273 (pCMV-Neo-Bam- p53 R273H), ZIKV NS2A (pOME0304), or KSHV orf10 (pOME0004) was measured with CellTiter-Glo luminescence cell viability assay kit following transfection with each vector (18h), treatment with indicated drugs (6h), and additional 24h-incubation. P-values calculated using Student t-test (n = 4) are shown as ‘ns’ (P>0.05), * (P≤0.05), **(P≤0.01), or *** (P≤0.001). (D-G) p53-Luc assays for ZIKV NS2A and KSHV orf10 expressing cells, untreated or stimulated with etoposide or nutlin-3. U2OS cells were transfected with either pGL3 control reporter (D) or p53-responsive reporter pGL13 (E-G) and EV, p53 R273, ZIKV NS2A-Flag, or KSHV orf10-Flag. At 18 hrs post transfection (p.t.), the cells were stimulated with 5 μM etoposide (F) or 10 μM nutlin-3 (G) for 6h or left untreated (D and E) and then incubated for 24h. Firefly luciferase levels were measured with One-Glo luciferase assay system (Promega Inc.).
Article Snippet: NLS-mCherry-NES reporter vector (pDN160) was a gift from Barbara Di Ventura & Roland Eils (Addgene plasmid # 72660; http://n2t.net/addgene:72660 ; RRID:Addgene_72660 [ ]). pOME0304, pOME0004, pOME0184, referred to as pDEST47-ZIKV NS2A-Flag, pDEST47-KSHV orf10-Flag, and pDEST47-KSHV orf57-Flag, respectively, express proteins fused to the C-terminal 3xFlag epitope ( ).
Techniques: Expressing, Reporter Assay, Western Blot, Luciferase, Transfection, Viability Assay, Plasmid Preparation, Incubation
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