PMC

p300 is involved in the PNR-mediated stimulation of p53 in HeLa cells. (A) Two different shRNA plasmids targeting p300 inhibited the PNR-mediated stimulation of p53RE-FLuc expression. The RLU in the PNR+shRNA-Con sample was normalized to 100%. (B) shRNA targeting p300 inhibits the PNR-mediated stimulation of p53 acetylation and accumulation. The indicated proteins were measured by immunoblotting. The numbers below the p300 band represent the levels of p300 protein normalized to the p300 level in sample 1. shRNA-Con, pLKO.1 empty lentiviral control.

PNR participates in a complex of p53 and p300 in HeLa cells. (A and B) Two days after cotransfection of HA-tagged PNR, p53, and p300, cells were lysed for immunoprecipitation with the indicated antibodies. Preimmune IgG and anti-His antibody were used as nonspecific binding controls. (A) Anti-p53 and anti-p300 antibodies coimmunoprecipitate (IP) HA-PNR. (B) Anti-HA antibody coimmunoprecipitates p53. (C and D) p53 and PNR colocalize in nuclei. Subcellular localizations of p53 and PNR were visualized by immunofluorescence (IF). Green, p53; red, HA; blue, DAPI. (C) Sample 1, p53 transfection; 2, PNR transfection; 3, cotransfection of PNR and p53. Arrow, colocalization of PNR and p53. (D) Enlarged image of a representative nucleus from a field of cells cotransfected with PNR and p53.

PNR enhances formation of a complex consisting of p53 and p300 with or without actinomycin D treatment in HeLa cells. (A) PNR enhances p300's binding to total p53 and Ac-p53. Anti-p300 antibody coimmunoprecipitated p53. Both p53 and Ac-p53 (K373 + 382) were measured by immunoblotting. Upper arrow, Ac-p53; lower arrow, rabbit IgG heavy chain. (B) PNR boosts actinomycin D-enhanced association between p53 and p300. One day after cotransfection of the indicated plasmids, cells were treated with 10 nM actinomycin D for 24 h and then processed as described for panel A.

PNR-stimulated p53 acetylation correlates with p53 transactivation and accumulation in HeLa cells. (A) PNR stimulates p53 acetylation. Total p53 and acetylated p53 (Ac-p53) levels were measured by immunoblotting with anti-p53 and anti-Ac-p53 antibodies, respectively. Acetylations by the indicated acetyltransferases, which were measured with their specific anti-Ac-p53 antibodies, can occur at multiple lysines of p53. The numbers below each band represent the levels of Ac-p53 per unit of total p53 normalized to that in sample 2. GFP was used as a transfection control. (B and C) Coexpressing p53 deacetylase SIRT1 inhibits PNR-mediated stimulation of p53 accumulation and transactivation. (B) SIRT1 inhibits PNR-mediated p53 acetylation and accumulation in a dose-dependent fashion. Ac-p53, total p53, PNR, and GFP were measured as described for panel A. Endogenous p21 and β-actin protein levels were also measured. SIRT1 abolished the PNR-mediated increase in p21 protein accumulation. (C) SIRT1 inhibited PNR-mediated stimulation of p53RE-FLuc expression in a dose-dependent fashion in a 96-well plate.

PNR-mediated stimulation of p53-responsive promoters requires p53. (A) In p53-null H1299 cells, PNR alone fails to stimulate p53RE-FLuc whereas PNR in the presence of exogenous p53 stimulates p53RE-FLuc. The RLU in the sample with 0 μg of p53 and PNR was normalized to 1. (B) shRNAs targeting p53 abolish PNR-mediated stimulation of p53RE-FLuc in HeLa cells. shRNA-GFP-expressed shRNA targeting GFP was used as a control. The RLU in the sample with both shRNA-GFP and PNR was normalized to 100%. Upper panel: immunoblot showing β-actin loading control and shRNA-mediated reductions in p53 protein accumulation. The numbers below each band represent the level of p53 protein normalized to β-actin and to the p53 level in sample 1. Lower panel: reporter assay. (C) shRNAs targeting p53 significantly inhibit PNR-mediated increases of p21 mRNA levels in HeLa cells. The p21 mRNA level was measured by qRT-PCR normalized to the sample with only shRNA-GFP transfected. (D) Mutation of p53-responsive elements abrogates PNR-mediated stimulation of MT-p53-RE-FLuc in HeLa cells. The RLU in the sample with only p53RE-FLuc was normalized to 1. (E) Dominant-negative mutant p53-C135Y abrogates the PNR-mediated stimulation of p53RE-FLuc in HeLa cells. The RLU in the sample with only p53RE-FLuc was normalized to 1.

PNR modulates p53 posttranslationally in HeLa cells. (A) PNR does not stimulate p53 transcription. Two days after transfection, p53 mRNA levels were measured by qRT-PCR and normalized to that of the sample with 0 μg of PNR. (B) PNR stabilizes p53 protein. Levels of endogenous p53 remaining after treatment with cycloheximide (2 μg/ml) for the indicated times were measured by immunoblotting, and the level in the 0-min sample was normalized to 1. Upper panel: GFP control transfection (1.2 μg). Middle panel: PNR transfection (1.2 μg). Lower panel: plot of remaining p53 protein levels versus treatment time. (C) PNR stimulates the specific activity of p53 as a transcriptional factor. Increasing amounts of the p53 expression plasmid were cotransfected with a constant amount of p53RE-FLuc to provide standard curves of p53 protein levels versus reporter activity. For both p53 protein measurement by immunoblotting (upper panel) and p53 transcriptional activity measurement by reporter assay (lower panel), the results determined under the conditions represented by sample 1 were normalized to 1. The middle panel shows the relative intensities of p53 immunoblotting signal (y axis) versus doses of p53 plasmid transfected (x axis). Sample numbers correspond to the conditions shown in the upper panel (sample 1, empty vector control; sample 2, PNR; samples 3 to 7, p53 standard curve). *, P < 0.05 (one-tailed test).

p53 enhances PNR-induced cell apoptosis. (A) PNR induces HeLa cell apoptosis in a dose-dependent fashion. Two days after transfection, an annexin V binding assay was used to quantitate apoptotic cells by flow cytometry. Pitx2a was used as a positive control (). The fraction of apoptotic cells in the sample with 0 μg of PNR was normalized to 1. Data representing the results of three repeated experiments are plotted on the right. (B) shRNAs targeting p53 significantly inhibit PNR-induced HeLa cell apoptosis. shRNA-Con, pLKO.1 empty expression vector control. The right plot shows relative levels of apoptosis based on the means of data determined under each set of conditions in 3 independent experiments. The mean for samples with only shRNA-Con was normalized to 1. *, P < 0.05 (one-tailed test). (C) p53 protein levels in three isogenic HCT116 cell lines containing two p53 alleles, one allele, or none. (D and E) PNR induces cell apoptosis in both p53^+/+ and p53^−/− HCT116 cells. Two days after transfection of PNR expression plasmid, the isogenic HCT116 cells were processed for both an annexin V binding assay and sub-G₁ analysis. The data from three repeated experiments is plotted on the right. (D) Annexin V binding assay. PNR mildly induced apoptosis at similar levels in both cell lines. (E) Sub-G₁ analysis. PNR induced apoptosis in p53^+/+ HCT116 cells at a level ∼2-fold higher than the level seen in p53^−/− HCT116 cells. PNR also increased apoptosis at the sub-G₁ phase in HeLa cells.

PNR enhances accumulation of p53 protein in HeLa cells and selectively stimulates p53-responsive promoters in both HeLa cells and HCT116 cells. (A) PNR increases levels of p53 and firefly luciferase fusion protein (p53/FLuc) in a dose-dependent fashion in HeLa cells. A p53/FLuc-IRES-RLuc reporter plasmid expressing the p53/FLuc fusion protein and Renilla luciferase as an internal control was cotransfected with the indicated amounts of PNR expression plasmid into HeLa cells in a 96-well plate. Two days after transfection, the luciferase activities were assayed. RLU, relative luciferase activity; FLuc, firefly luciferase; IRES, internal ribosome entry site; Rluc, Renilla luciferase. (B) PNR enhances accumulation of endogenous p53 in HeLa cells. p53 levels were measured by immunoblotting (IB) 2 days after transfection of PNR expression plasmid into HeLa cells in 6-cm-diameter dishes. The ratio of p53 signal to β-actin signal in the sample with 0 μg of PNR was normalized to 1. *, P < 0.05 (one-tailed test). (C) PNR stimulates p53RE-FLuc, a p53-responsive firefly luciferase reporter plasmid, in a dose-dependent fashion in HeLa cells. Cells were cotransfected with the indicated amounts of a PNR expression plasmid and other plasmids in a 96-well plate. Two days later, the luciferase activities were assayed and normalized to the level in the sample with 0 μg of PNR. (D) Endogenous PNR contributes to p53 response in HeLa cells. Cells in a 96-well plate were cotransfected with 0.02 μg of p53RE-FLuc and the indicated shRNA expression plasmids targeting p53 and PNR. shRNA-Con, pLKO.1 empty lentiviral control. The FLuc activity in the sample with shRNA-Con was normalized to 100%. *, P < 0.05 (two-tailed test). (E, F, and G) PNR selectively stimulates p53 target genes in HeLa cells and p53^+/+ HCT116 cells. Cells were transfected with the indicated amounts of the PNR expression plasmid (E and F) or shRNA expression plasmids (G) in 6-cm-diameter dishes. Two days later, mRNA levels of the indicated p53 target genes were measured by qRT-PCR and normalized to β-actin or RPL38 mRNA levels. For each gene, the mRNA level in the sample with 0 μg of PNR (E and F) or shRNA-GFP (G) was normalized to 1. (E) PNR overexpression in HeLa cells. (F) PNR overexpression in HCT116 cells. (G) Knockdown of endogenous PNR in HeLa cells. Sample 1, shRNA-GFP; sample 2, shRNA-PNR-A; sample 3, shRNA-PNR-B; sample 4, shRNA-p53.