Article Figures & Data
Figures
- Figure 1.
Inhibition or overexpression of NQO2 and NQO1 leads to alterations in stability of p53. A, Western analysis. Human hepatoblastoma Hep-G2 cells in left three panels were transfected with indicated amounts of siNQO2 RNA. The lane showing 0 siNQO2 was transfected with 80 nmol/L control scrambled siRNA. The Hep-G2 cells in right-most panel were transfected with increasing concentration of Flag-NQO2 expression plasmid. The lane showing 0.0 was transfected with 4.0 μg of control Flag vector. The transfected cells were lysed. The lysates were separated on SDS-PAGE, Western blotted and probed with NQO2, NQO1, p53, p27, p21, Flag, and actin antibodies. B, Western analysis. Hep-G2 cells were transfected with increasing concentrations of siNQO1 RNA (left) or pcDNA-NQO1-V5 plasmid (right). The cells were lysed, Western blotted, and probed with p53, NQO1, V5, and actin antibodies. C, p53-luciferase assay. Left, Hep-G2 cells were cotransfected with a fixed concentration of pFc-p53 expression, p53-luciferase (p53-Luc), and renilla luciferase plasmids and an increasing concentration of siNQO2 or siNQO1 RNA, lysed and analyzed for luciferase activity. The control lane (C) shows fold increase in p53-Luc expression in cells overexpressing p53 from pFc-p53 plasmid compared with vector transfected control. Right, Hep-G2 cells were cotransfected with increasing concentration of pcDNA-NQO2 or pcDNA-NQO1 expression plasmid and fixed concentrations of p53-luciferase and renilla luciferase, lysed and analyzed for luciferase activity. D, apoptotic cell death. Hep-G2 cells were transfected with indicated concentrations of NQO2 or NQO1 siRNA, incubated for 72 h, treated with Annexin V, and analyzed for apoptotic cells by flow cytometry.
- Figure 2.
Proteasome inhibitors stabilize p53 in NQO2 and NQO1-null cells. A, degradation of p53 in wild-type, NQO2-null, and NQO1-null keratinocytes. Wild-type, NQO1-null, and NQO2-null keratinocytes were treated with 5 μmol/L MG132 for 0–8 h or 50 μmol/L lactacystin for 6 h. The concentrations and time used for MG132 and lactacystin treatments did not lead to cell death as determined by visual observations under the microscope and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of viability test (data not shown). Cells were lysed in ice-cold RIPA. Fifty micrograms of protein were loaded in each lane and separated on SDS polyacrylamide gels, blotted on the ECL membranes, and probed with antibodies against tumor suppressor proteins p53 and actin. B, degradation of p53 in wild-type, NQO2-null, and NQO1-null mouse macrophages. Wild-type, NQO2-null, and NQO1-null mouse macrophages were treated with MG132 for 5 h, washed and treated with cycloheximide. The cells were collected at varying time intervals, lysed, and analyzed by Western blotting and probing with p53, NQO2, NQO1, and actin antibodies. The experiments in B were done thrice, and results were reproducible. Representative experiments.
- Figure 3.
NQO2 and NQO1 interaction with p53. A, immunoprecipitation. Hep-G2 cells were transfected with Flag vector or Flag-NQO2 expression plasmid. The NQO2-Flag transfected cells were lysed and immunoprecipitated with anti-Flag and IgG antibodies and analyzed by Western blotting and probing with anti-Flag and anti-p53 antibodies. B, copurification of p53 with NQO1. Wild-type and NQO1-null keratinocytes were lysed with cold RIPA and protease inhibitor cocktail. Lysates were passed through dicumarol column. Columns were washed by passing excess amount of RIPA. NQO1 protein was then eluted, separated on polyacrylamide gels, blotted on the ECL membrane, and probed with antibodies against p53 and NQO1. The last two lanes were loaded with 50 μg of total cell lysate (TCL) of wild-type and NQO1-null keratinocyte cells. C, SEAP assay. Mouse hepatoma Hepa-1 cells were transfected with the plasmids in combinations: control (pM+pVP16+pG5SEAP); p53 control (pM-p53+pVP16+pG5SEAP); NQO1 control (pM+pVP16-mNQO1+pG5SEAP); NQO2 control (pM+pVP16-mNQO2+pG5SEAP); NQO2 interaction with p53 experiment (pM-p53+pVP16-mNQO2+pG5SEAP); NQO1 interaction with p53 experiment (pM-p53+pVP16-mNQO2+pG5SEAP); positive control (pM3-VP16+pG5SEAP, which have vectors encoding interacting proteins). The SEAP activity in culture supernatant was determined using a BD Great EscAPeTM SEAP chemiluminescence detection kit (Clontech) according to the manufacturer's instruction. Columns, mean of five independent experiments; bars, SD.
- Figure 4.
NQO2 and NQO1 interaction with p53 and protection against 20S proteasomal degradation of p53. A, Western analysis of purified 20S proteasomal fractions. NQO2, NQO1, and p53 copurify with 20S proteasomes. Livers from wild-type, NQO2-null, and NQO1-null mice were homogenized and subcellular fractionated. The 20S-containing fraction was purified and analyzed for the presence of p53, NQO1, NQO2, 26S, and actin. The proteins were separated on SDS-PAGE, Western blotted, and probed with rabbit polyclonal anti-20S proteasome core subunits, rabbit polyclonal anti-p53, rabbit–anti-NOQ1, goat–anti-NQO2, rabbit–anti-26S proteasome, S6-subunit, mouse–anti-actin antibodies. B, immunoprecipitation of liver 20S fractions. Two hundred micrograms of 20S proteasomes purified from livers of wild-type, NQO2-null, and NQO1-null mouse were immunoprecipitated with rabbit–anti-20S antibody or rabbit–anti-IgG antibody. The proteins were separated on SDS-PAGE, Western blotted, and probed with 20S, NQO2, NQO1, p53, and actin antibodies. Input lanes contain one-tenth of amount of 20S fraction used for immunoprecipitation. C, immunoprecipitation of liver cytosolic fractions. One milligram liver cytosolic proteins from wild-type, NQO1-null, and NQO2-null mice was immunoprecipitated with rabbit–anti-20S antibody or rabbit–anti-IgG antibody. The proteins were separated on SDS-PAGE, Western blotted, and probed with 20S, NQO2, NQO1, p53, and actin antibodies. Input lanes contain one-tenth of amount of cytosolic fractions used for immunoprecipitation. D, in vitro degradation of p53. NQO2 and NQO1 protection against 20S degradation of p53. In vitro reticulocyte lysate-translated [35S]methionine-labeled p53 was incubated with 2 μg of purified 20S proteasome at 37° for 1 h in the absence or presence of in vitro translated NQO2 or NQO1 together with or without 1 mmol/L NADH or 500 μmol/L NRH. Samples were mixed with Laemmli denaturation buffer, heated at 95°C for 5 min, and electrophoresed on SDS-PAGE. After electrophoresis, the gel was dried and exposed overnight at −70° and autoradiographed. *, unspecific band.
- Figure 5.
NQO2 and NQO1 protection against 20S proteasomal degradation of p53 in temperature-sensitive ubiquitin-deficient cells and effect of radiation. A, Western analysis. Expression of p53 in A31N-ts20 cells at permissive and restrictive temperatures. A31N-ts20 cells were cultured at 35°C (permissive temperature, ubiquitin positive) and 39°C (restrictive temperature, ubiquitin negative). The cells grown at 39°C were exposed to 4 Gy of γ radiation, lysed in RIPA, and analyzed by Western blotting and probing with anti-p53 and antiactin antibodies. B, siRNA transfection and Western analysis. A31N-ts20 cells were transiently transfected with HA-tagged p53 without and with increasing concentrations of NQO2-siRNA (left), NQO1-siRNA (middle), or control siRNA (right) at 35°C. The cells were incubated for 48 h at 35°C and then transferred to 39°C for 24 h. The cells were either unexposed or exposed to 4-Gy γ radiation, and cultured at 39°C for another 4 h. The cells were lysed and analyzed by Western blotting and probing with anti-NQO2, anti-NQO1, anti-p53, and antiactin antibodies. C, NQO2 and NQO1 siRNA combined transfection and Western analysis. NQO2 siRNA and NQO1 siRNA were combined in concentrations and experiments repeated as in B.
- Figure 6.
Chemical induction of NQO2 and NQO1 and protection against 20S degradation of p53. A, Western analysis. Benzo(a)pyrene induction of NQO2 and NQO1 and stability of p53. Wild-type and NQO2-null keratinocytes (left) and wild-type and NQO1-null keratinocytes (right) were treated with 1,200 nmol benzo(a)pyrene in acetone. Control cells received acetone alone. The cells were harvested and homogenized in appropriate buffer containing protease inhibitors. One hundred micrograms of homogenates were separated on SDS-PAGE, Western blotted, and probed with anti-NQO2, anti-NQO1, anti-p53, and antiactin antibodies. B, hypothetical model demonstrating the role of NQO1 and NQO2 in protection against 20S proteasomal degradation of p53.
Volume 67, Issue 11
