The pCMV-SP6-PNR plasmid expressing PNR was constructed by subcloning a full-length wild-type PNR into a pCMV-SP6 expression vector from pcDNA3.1/HisC-PNR (31
), kindly provided by S. M. Chen (Washington University). The pCMV-SP6-HA-PNR plasmid expressing N-terminally hemagglutinin (HA)-tagged PNR (see and ) was constructed by adding an HA tag coding sequence to the 5′ terminus of PNR with no space. Reporter plasmid p53RE-FLuc, expressing firefly luciferase from a p53-responsive promoter containing two tandem p53-responsive elements, was from Panomics (catalog no. LR0057). A p53RE-FLuc derivative with the p53 binding site inactivated was generated by mutating critical CXXG residues (7
) into AXXT with a QuikChange II XL site-directed mutagenesis kit (Agilent catalog no. 200521). The primers used for this mutation were 5′-CGC GTG CTA GCT ACA GAA aAT t
TC TAA GaA Tt
C TGT GCC TTG CCT GGA aTT t
CC TGG CaT Tt
C CTT GGG AGA TCT GGG TAT-3′ and 5′-ATA CCC AGA TCT CCC AAG GaA At
G CCA GGa AAt
TCC AGG CAA GGC ACA GaA Tt
C TTA GAa ATt
TTC TGT AGC TAG CAC GCG-3′, where the lowercase letters represent mutated nucleotides. A plasmid expressing human p53 dominant-negative mutant p53C135Y
was from Clontech (catalog no. 631922). A pCMV-SP6-Pitx2a plasmid expressing Pitx2a was constructed by subcloning full-length wild-type Pitx2a into a pCMV-Sp6 expression vector from a green fluorescent protein-Pitx2a (GFP-Pitx2a) plasmid (50
), kindly provided by Q. Z. Wei (Kansas State University).
Fig 7 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 (more ...)
Fig 8 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) (more ...) Cell culture.
HeLa cells (ATCC catalog no. CCL-2), p53+/+ and p53−/− HCT116 cells (kindly provided by B. Vogelstein, John Hopkins University), and p53-null H1299 cells (a gift from W. Sugden, University of Wisconsin—Madison) were cultured at 37°C in Dulbecco's modified Eagle medium (DMEM)–10% fetal bovine serum (FBS) (heat inactivated) in a 5% CO2 atmosphere.
Luciferase reporter assays.
Luciferase reporter assays were performed using 96-well plates and reverse transfection (RT). For each well, 0.01 μg of reporter plasmid p53RE-Fluc, 0.005 μg of phRL-SV40 expressing Renilla luciferase, and the indicated amounts of PNR expression plasmid and other plasmids were added to a total of 0.085 μg of DNA in 5 μl of Opti-MEM. TransIT-LT1 (0.17 μl) was mixed with 9 μl of Opti-MEM, mixed with the DNA as described above, transferred to the microplate, and incubated for 30 min at room temperature. A total of 1.2 × 104 cells in 100 μl of medium were then added per well. Two days after transfection, cells were processed using a Dual-Glo luciferase assay (Promega catalog no. E2940) and a Perkin-Elmer luminometer.
For an annexin V binding assay, 106 cells were seeded per 6-cm-diameter dish and incubated for 18 h at 37°C. Equal amounts of total plasmids were then cotransfected using Lipofectamine 2000 (Invitrogen catalog no. 11668-019) (2:1 Lipofectamine:DNA solution) following the manufacturer's protocol. The medium was changed 6 h after transfection. Two days after transfection, a Vybrant 2 Alexa Fluor 488 apoptosis assay kit (Invitrogen catalog no. V13241) was used to label apoptotic cells according to the manufacturer's instructions. Flow cytometry was used to count the labeled cells, and the data were analyzed using Flowjo software. The percentages of apoptotic cells (see ) represent the proportions of Alexa Fluor 488-labeled cells among the transfected cells expressing red fluorescent protein.
Fig 3 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 (more ...) Sub-G1 apoptosis assay.
Cells in 6-cm-diameter dishes were transfected with Lipofectamine 2000 as described above, except that the indicated plasmids were used. Two days after transfection, cells were detached, treated as described elsewhere (http://sciencepark.mdanderson.org/fcores/flow/files/DNA_PI.html
), and measured by flow cytometry.
Cells in 6-cm-diameter dishes were transfected with Lipofectamine 2000 as described above, except that the indicated plasmids were cotransfected. Cells were harvested 2 days after transfection. After two washes with cold phosphate-buffered saline (PBS), the cell pellet was lysed with 200 μl of radioimmunoprecipitation assay (RIPA) buffer (Pierce catalog no. 89900) plus protease inhibitors (Roche catalog no. 11873580001) for 15 min on ice. A 50-μl volume of 5× sodium dodecyl sulfate (SDS) sample buffer was added. The sample was sonicated twice for 5 s each time with a probe sonicator at 4°C and heated for 15 min at 99°C with vigorous vortex mixing. After centrifugation at 9,200 × g for 10 min at 4°C, the supernatant was stored at −20°C.
Cell lysates were subjected to electrophoresis on a freshly made 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes (GE catalog no. RPN303D) in transfer buffer with 10% methanol, except that 5% SDS-polyacrylamide gel electrophoresis (5% SDS-PAGE) was used to resolve p300. The membrane was blocked with Odyssey blocking buffer (LiCor catalog no. 927-40000) for 1 h at room temperature, incubated with primary antibodies for 1 h at room temperature, and washed 5 times for 5 min each time with PBS–0.1% Tween 20. For semiquantitative analysis, the membrane was then incubated with fluorescent secondary antibodies (LiCor goat anti-rabbit antibody conjugated with IRDye 800cw [catalog no. 926-32211] and goat anti-mouse antibody conjugated with IRDye 680 [catalog no. 926-32220]) for 40 min at room temperature, followed by 5 washes performed as described above. The air-dried membranes bearing a dilution curve of each sample were scanned using a LiCor Odyssey imager, and images were analyzed using Odyssey version 3.0 software. For detection of HA-tagged PNR (see ), samples were transferred to polyvinylidene difluoride (PVDF) membranes in transfer buffer with 15% methanol, a rat anti-HA antibody conjugated to horseradish peroxidase (HRP; Roche catalog no. 12013819001) was used as the primary antibody, and the secondary antibody was omitted.
For immunoblotting, anti-acetylated p53 antibodies (K373 + 382 [Upstate catalog no. 06-758], K382 [Upstate catalog no. 04-1146], K320 [Upstate catalog no. 06-1283], and K120 [AbCam catalog no. ab78316]) (1:1,000), mouse anti-total p53 antibody (Calbiochem catalog no. OP43) (1:1,000), rabbit anti-PNR antibody (Sigma catalog no. P5373) (1:1,000), rabbit anti-p300 antibody (Santa Cruz catalog no. sc-584) (1:1,000), rabbit anti-β-actin antibody (Santa Cruz catalog no. sc-1616-R) (1:3,000), mouse anti-GFP antibody (Covance catalog no. MMS-118R) (1:6,000), and LiCor fluorescent secondary antibodies (1:10,000) were diluted in Odyssey blocking buffer as indicated and mouse anti-p21 antibody (Santa Cruz catalog no. SC-56335) (1:500), HRP-conjugated rat anti-HA antibody (1:2,000), HRP-conjugated goat anti-mouse secondary antibody (1:10,000), and HRP-conjugated mouse anti-rabbit light chain secondary antibody (1:20,000) were diluted in PBS–0.1% Tween 20–5% nonfat milk as indicated.
Cells in 6-cm-diameter dishes were transfected with Lipofectamine 2000 as described above, except that 0.5 μg of p53-expressing plasmid, 1.5 μg of HA-PNR-expressing plasmid, and 1.5 μg of p300-expressing plasmids were cotransfected. Two days after transfection, cells were detached and washed 3 times with cold PBS. Cell pellets were resuspended with 400 μl of buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM PMSF) and incubated for 15 min on ice. A 25-μl volume of 10% NP-40 was added, followed by vigorous vortex mixing for 10 s. The lysate was centrifuged at 2,300 × g for 1 min at 4°C. The supernatant (cytoplasmic extract) was kept on ice. The nuclear pellet was resuspended in 200 μl of buffer B (20 mM HEPES [pH 7.9], 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5 mM PMSF) and sonicated twice for 5 s each time. The resulting nuclear lysate was subjected to vigorous vortex mixing for 15 min and centrifuged at 13,000 × g for 10 min at 4°C. The supernatant (nuclear extract) was combined with the cytoplasmic extract for immunoprecipitation.
Two micrograms of rabbit antibodies against p53 (Santa Cruz catalog no. sc-6243) or p300 (Santa Cruz catalog no. sc-584) or HA (Sigma catalog no. H6908) was mixed with 50 μl of protein A Dynabeads (Invitrogen catalog no. 100-01D). Two micrograms of rabbit anti-His antibody (catalog no. sc-803) and 2 μg of preimmune rabbit IgG (catalog no. sc-2344) from Santa Cruz were used as controls. Immunoprecipitation was performed following the manufacturer's instructions, except that the Dynabeads were washed 6 times for 30 s each time after immunoprecipitation. Dynabeads were then incubated with 100 μl of 1× SDS sample buffer for 20 min at 50°C with vigorous vortex mixing to elute proteins. The beads were centrifuged at 9,200 × g for 1 min at 4°C, and the supernatant was stored at −20°C after 10 min at 99°C.
Cells were reverse transfected described above in “Luciferase reporter assays,” except that 8-chamber slides were used and all material volumes were doubled for each chamber. Two days after transfection, the cells were washed 3 times with PBS and fixed in 1% paraformaldehyde–PBS for 30 min at room temperature. Cells were washed with PBS 3 times and permeabilized in 0.5% Triton X-100–PBS for 30 min at room temperature. After another 3 washes, the cells were blocked in PBS containing 5% normal horse serum and DAPI (4′,6-diamidino-2-phenylindole) (5 μg/ml) for 5 to 6 h at 4°C. Subsequently, cells were washed 4 times and rabbit anti-p53 antibody (Santa Cruz catalog no. sc-6243) (1:200) and mouse anti-HA antibody (Roche catalog no. 11583816001) (1:100) were added to PBS containing 5% normal horse serum to cover the cells for 8 h at 4°C. The cells were washed 4 times and incubated with goat anti-mouse Alexa Fluor 568-conjugated antibody (Invitrogen catalog no. A11004) (1:1,000) and goat anti-rabbit Alexa Fluor 488-conjugated antibody (Invitrogen catalog no. A31627) (1:1,000) in PBS containing 3% normal horse serum for 1 h at room temperature. After 4 washes, the slide was briefly dried, 40 μl of mounting medium containing DAPI (Vector Laboratories catalog no. H-1200) was added, and the slide was covered with a coverslip for imaging with a confocal microscopy.
Reverse-transcription real-time PCR.
Cells were reverse transfected as described for the reporter assays above, except that 6-cm-diameter dishes were used and all material volumes were increased by 60-fold. Two days after transfection, total RNA was extracted using an RNeasy minikit (Qiagen catalog no. 74106) and treated using DNase for 30 min at 37°C. A 0.1-μg volume of total RNA was added to each reaction mixture.
TaqMan One-Step RT-PCR Master Mix reagents (Applied Biosystems catalog no. 4309169) were used for reverse transcription real-time PCR with a 7900HT real-time PCR system (Applied Biosystems) programmed for 48°C for 30 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. mRNA levels of β-actin or RPL38 were used as internal controls. The ratio of the TaqMan primer to the probe was fixed at 2:1. The final probe concentration in a 20-μl reaction volume per well in a 96-well plate was approximately 50 to 100 nM. The primer and probe sets (listed here as forward primer, reverse primer, and probe) were as follows: for p53, 5′-TTTCCGTCTGGGCTTCT-3′, 5′-TGGAATCAACCCACAGCT-3′, and 5′–6-carboxyfluorescein (FAM)/TGTGACTTGCACGTACTCCCCTG/IBFQ-3′; for p21, 5′-TTCCTGTGGGCGGATTA-3′, 5′-GAGCAGGCTGAAGGGT-3′, and 5′-FAM/CGTTTGGAGTGGTAGAAATCTGTCATGC/IBFQ-3′; for Puma, 5′-GAGATGGAGCCCAATTAGGTG-3′, 5′-ACATGGTGCAGAGAAAGTCC-3′, and 5′-FAM/AGGGTGTCAGGAGGTGGGAGG/IBFQ-3′; for MDM2, 5′-TGCCAAGCTTCTCTGTGAAAG-3′, 5′-TCCTTTTGATCACTCCCACC-3′, and 5′-FAM/ACCTGAGTCCGATGATTCCTGCTG/IBFQ-3′; for Pirh2, 5′-GGTCAAGAGCGAGGTCAG-3′, 5′-CACAAGCGGCAAGTATAAAGC-3′, and 5′-FAM/ACAGCAAGGTGCCTTTAGGAGACATC/IBFQ-3′; for PNR, 5′-GGGAAGCACTATGGCATCTATG-3′, 5′-CACCTGGCACCTGTAGATG-3′, and 5′-FAM/CGCCGTACGCTCCTCTTGAAGAA/IBFQ-3′; for RPL38, 5′-GCAGATACCTTTACACCCTGG-3′, 5′-CTGGTTCATTTCAGTTCCTTCAC-3′, and 5′-FAM/TGCTTCAGTTTCTCTGCCTTCTCTTTGT/IBFQ-3′; and for β-actin, 5′-TCACCCACACTGTGCCCATCTACGA-3′, 5′-CAGCGGAACCGCTCATTGCCAATGG-3′, and 5′-FAM/ATGCCCTCCCCCATGCCATCCTGCGT/TAMRA-3′.
Protein stability assay.
Cells in 6-cm-diameter dishes were processed as described above for immunoblotting, except that the indicated plasmids were transfected and cells were treated with cycloheximide (Sigma catalog no. C4859) (2 μg/ml) for 0, 15, 30, or 60 min before being harvested for immunoblotting.
pLKO.1-based short hairpin RNA (shRNA) expression plasmids were cotransfected with the indicated plasmids and transfection reagents in the different assays as described above. Two days after transfection, cells were processed for assays. For each target gene, two shRNA plasmids were validated to efficiently knock down the indicated gene at two different fragments. shRNA expression plasmids targeting p53 (catalog no. RHS4533-NM_000546), PNR (catalog no. RHS4533-NM_014249), p300 (catalog no. RHS4533-NM_001429), GFP (catalog no. RHS4459), and pLKO.1 empty vector plasmid (catalog no. RHS4080) were from Open Biosystems.
P values were calculated using paired Student's t-tests.