February 2011
Volume 52, Issue 2
Free
Glaucoma  |   February 2011
Quercetin Induces the Expression of Peroxiredoxins 3 and 5 via the Nrf2/NRF1 Transcription Pathway
Author Affiliations & Notes
  • Naoya Miyamoto
    From the Departments of Molecular Biology,
    Ophthalmology, and
  • Hiroto Izumi
    From the Departments of Molecular Biology,
  • Rie Miyamoto
    Ophthalmology, and
  • Hiroyuki Kondo
    Ophthalmology, and
  • Akihiko Tawara
    Ophthalmology, and
  • Yasuyuki Sasaguri
    Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Fukuoka, Japan.
  • Kimitoshi Kohno
    From the Departments of Molecular Biology,
  • Corresponding author: Kimitoshi Kohno, Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan; k-kohno@med.uoeh-u.ac.jp
Investigative Ophthalmology & Visual Science February 2011, Vol.52, 1055-1063. doi:10.1167/iovs.10-5777
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      Naoya Miyamoto, Hiroto Izumi, Rie Miyamoto, Hiroyuki Kondo, Akihiko Tawara, Yasuyuki Sasaguri, Kimitoshi Kohno; Quercetin Induces the Expression of Peroxiredoxins 3 and 5 via the Nrf2/NRF1 Transcription Pathway. Invest. Ophthalmol. Vis. Sci. 2011;52(2):1055-1063. doi: 10.1167/iovs.10-5777.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: The flavonoids have potent antioxidant and free-radical scavenging properties and are beneficial in the prevention and treatment of ocular diseases including glaucoma. The authors have previously reported that antiglaucoma agents could transcriptionally activate the antioxidant protein peroxiredoxin (PRDX)2. The purpose of this study was to investigate whether quercetin can activate transcription factors and induce the expression of the PRDX family.

Methods.: To demonstrate whether quercetin can transcriptionally induce the expression of the PRDX family, trabecular meshwork cells were treated with quercetin, and PRDX expression and transcription factors were both investigated by Western blot analysis, reporter assays, and siRNA strategies. Subsequently, cellular sensitivity to oxidative stress was determined.

Results.: Expression of the PRDX3 and PRDX5 genes was induced by quercetin in a time- and dose-dependent manner. NRF1 transactivates the promoter activity of both PRDX3 and PRDX5 but not PRDX2 and PRDX4. Quercetin can also induce the expression of Nrf2 and NRF1 but not of Ets1, Ets2, or Foxo3a. Knockdown of NRF1 expression significantly reduced the expression of both PRDX3 and PRDX5. Reporter assays showed that NRF1 transactivated the promoter activity of both PRDX3 and PRDX5 and that the downregulation of NRF1 with siRNA repressed the promoter activity of both PRDX3 and PRDX5. Furthermore, the downregulation of NRF1, PRDX3, and PRDX5 renders trabecular meshwork cells sensitive to hydrogen peroxide. Finally, NRF1 activation by quercetin was completely abolished by the knockdown of Nrf2.

Conclusions.: Quercetin upregulates the antioxidant peroxiredoxins through the activation of the Nrf2/NRF1 transcription pathway and protects against oxidative stress-induced ocular disease.

Flavonoids such as quercetin (3,5,7,3′,4′-pentahydroxy flavone) can protect cells from oxidative stress. 1 4 Quercetin—present in fruit, vegetables, and many other dietary sources—is one of the most widely distributed flavonoids. 5 It has been shown that certain flavonoids can induce antioxidant responsive element-dependent gene expression through the activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). 6 Oxidative stress plays an important role in the pathogenesis of multiple ocular diseases, including glaucoma. 7  
Glaucoma is a major cause of irreversible blindness worldwide and is characterized by cupping of the optic nerve head and irreversible loss of retinal ganglion cells. 8 Elevated intraocular pressure (IOP) caused by a reduction in aqueous outflow is a major risk factor in the development of glaucoma 9 and the progression of glaucomatous damage to the optic nerve. 10 12 The trabecular meshwork (TM) is a reticulated tissue at the iridocorneal junction that makes intimate contact in the juxtacanalicular region with the canal of Schlemm for aqueous humor filtration. 13 Oxidative stress is reported to trigger degeneration in the human TM and its endothelial cell components, subsequently leading to an increase in IOP and glaucoma. Increasing evidence indicates that reactive oxygen species (ROS) play a key role in the pathogenesis of glaucoma. 14 17  
Peroxiredoxins (PRDXs) are a family of enzymes that catalyze the reduction of hydrogen peroxide. 18 22 There are five 2-Cys types that contain two conserved cysteine residues. These PRDXs are expressed in a wide variety of cell types. However, the precise mechanisms controlling PRDX expression are not well understood. We have previously shown that oxidative stress can induce PRDX1 and PRDX5 through activation of the Ets1 transcription factor. 23 Furthermore, we have reported that antiglaucoma agents transcriptionally upregulate the PRDX2 gene through the activation of Foxo3a. 24 Thus, several transcription factors can regulate each PRDX gene. Here, we investigated whether quercetin induces gene expression of PRDX3 and PRDX5 through the Nrf2/NRF1 transcription pathway. 
Experimental Procedures
Cell Culture
The immortalized TM cell line, NTM5, derived from a normal trabecular meshwork, was kindly provided by Abott F. Clark (Glaucoma Research, Alcon Research, Ltd., Fort Worth, TX) and was cultured in Dulbecco's modified Eagle's medium (Nissui Seiyaku Co., Tokyo, Japan). 24,25 The primary TM cell (HTMC) was purchased from Sciencell Research Laboratories (San Diego, CA) 
Antibodies and Drugs
Antibodies against FKHRL1 (Foxo3a) (sc-9812), Ets1 (sc-111), Ets2 (sc-351), Nrf2 (sc-30915), PCNA (sc-56), PRDX2 (sc-23967), and PRDX4 (sc-23974) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti–β-actin antibody (AC-15) was purchased from Sigma. Generation of antibodies against PRDX1 and PRDX5, 23 NRF1, 26 and mitochondrial transcription factor A (mtTFA) 27 has been described previously. The anti–PRDX3 antibody was a kind gift from Hiroki Nanri (Seinan Jogakuin University, Kyushu, Japan). 28 Quercetin dihydrate was purchased from Sigma-Aldrich Co. (St. Louis, MO). Drug concentrations in this study corresponded with those used in clinical practice. 
Plasmid Construction
To obtain full-length cDNAs for human NRF1, PCR was carried out on a cDNA library (SuperScript; Invitrogen Life Technologies, Carlsbad, CA) using the following primer pairs (underlining indicates the start codon and stop codon): 5′-ATGGAGGAACACGGAGTGACCC-3′ and 5′-TCACTGTTCCAATGTCACCACCTCC-3′. The resultant PCR product was cloned (pGEM-T Easy Vector; Promega, Madison, WI). To construct a plasmid expressing Flag-tagged NRF1, N-terminal Flag-tagged NRF1 cDNA was ligated into the pcDNA3 vector (Invitrogen). The luciferase (Luc) constructs PRDX2-Luc (−402 to +68), PRDX3-Luc (−357 to +42), PRDX4-Luc (−306 to +36), and PRDX5-Luc (−314 to +113) have been described previously. 23 The following primer pairs were used: 5′-AGATCTTAGATGCTGCAGCCTCAGC-3′and 5′-AAGCTTGGCAAAGGCTAGACGCACGG-3′ for PRDX2-Luc; 5′-AGATCTTAGCTTATTAACGGACTAAAAC-3′ and 5′-AAGCTTCAGTGCACTCGGGCGCCACGG-3′ for PRDX3-Luc; 5′-AGATCTGTGAGGGGCTTGTGTGCAG-3′ and 5′-AAGCTTCACGCGAGCGCAGAAACACG-3′ for PRDX4-Luc; and 5′-AGATCTAAGATGCAAATCATATGC-3′ and 5′-AAGCTTCCCACGGCCACTTCCACTCC-3′ for PRDX5-Luc. 
Figure 1.
 
(A) Effect of quercetin on PRDX expression. NTM5 cells were incubated with 1 μM quercetin for the times indicated. Whole cell lysates (50 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. (B) NTM5 cells were cultured for 12 hours in the control medium or medium containing the indicated concentrations of quercetin. Whole cell lysates (50 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. (C) Primary HTMCs were incubated with 1 μM quercetin for the times indicated. Whole cell lysates (50 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue.
Figure 1.
 
(A) Effect of quercetin on PRDX expression. NTM5 cells were incubated with 1 μM quercetin for the times indicated. Whole cell lysates (50 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. (B) NTM5 cells were cultured for 12 hours in the control medium or medium containing the indicated concentrations of quercetin. Whole cell lysates (50 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. (C) Primary HTMCs were incubated with 1 μM quercetin for the times indicated. Whole cell lysates (50 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue.
Knockdown Analysis Using Small Interfering RNAs (siRNAs)
The following double-stranded RNA 25-bp oligonucleotides were commercially generated (Invitrogen): PRDX3 small interfering RNA (siRNA), 5′-UUUACCUUCUGAAAGUACUCUUUGG-3′ (sense) and 5′-CCAAAGAGUACUUUCAGAAGGUAAA-3′ (antisense); PRDX5 siRNA, 5′-AGAACCUCUUGAGACGUCGAUUCCC-3′ (sense) and 5′-GGGAAUCGACGUCUCAAGAGGUUCU-3′ (antisense); NRF1#1 siRNA, 5′-AUUAGACUCAAAUACAUGAGGCCGU-3′ (sense) and 5′-ACGGCCUCAUGUAUUUGAGUCUAAU-3′ (antisense); NRF1#2 siRNA, 5′-AUCUGAGUCAUCGUAAGAGGUGUCC-3′ (sense) and 5′-GGACACCUCUUACGAUGACUCAGAU-3′ (antisense); Nrf2 siRNA, 5′-AAUCACUGAGGCCAAGUAGUGUGUC-3′ (sense) and 5′-GACACACUACUUGGCCUCAGUGAUU-3′ (antisense). siRNA transfections were performed as described previously. 29,30 Briefly, 250 pmol of the indicated siRNA or control synthetic RNA (Stealth RNAi; Invitrogen) was transfected into 1 × 106 NTM5 cells; 1.5 × 105 cells were used for luciferase assays, and 2.5 × 103 cells were used for the WST-8 assay, as described. The remaining cells were subjected to Western blot analysis after 72-hour culture, as described. 
Western Blot Analysis
The preparation of whole cell lysates and whole nuclear lysates has been described previously. 29,30 The indicated amounts of whole cell lysate or whole nuclear lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride microporous membranes (Millipore, Billerica, MA) using a semidry blotter. The blotted membranes were treated with 5% (wt/vol) skimmed milk in 10 mM Tris, 150 mM NaCl, and 0.2% (vol/vol) Tween 20 and were incubated for 1 hour at room temperature with primary antibody. The following antibodies and dilutions were used: 1:500 dilution of anti-Nrf2, 1:5000 dilution of anti-PRDX2, 1:5000 dilution of anti-PRDX3, 1:1000 dilution of anti-PRDX4, 1:1000 dilution of anti-PRDX5, 1:1000 dilution of anti-Ets1, 1:1000 dilution of anti-Ets2, 1:5000 dilution of anti-Foxo3a, 1:5000 dilution of anti-NRF1, 1:5000 dilution of anti-mtTFA, 1:5000 dilution of anti-PCNA, and 1:20,000 dilution of anti–β-actin. Membranes were then incubated for 40 minutes at room temperature with a peroxidase-conjugated secondary antibody and were visualized using an enhanced chemiluminescence kit (GE Healthcare Bio-Science, Uppsala, Sweden), and membranes were exposed to Kodak film (X-OMAT; Kodak, Rochester, NY). For the correlation assay, the intensity of each signal was quantified using ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). 
Figure 2.
 
(A) Schematic representations of the PRDX luciferase constructs PRDX2-Luc, PRDX3-Luc, PRDX4-Luc, and PRDX5-Luc, with their CpG islands and transcription factor binding sites. (B) Transcriptional activity of the PRDX2-5 genes in response to quercetin treatment. The indicated reporter plasmids were transiently transfected into NTM5 cells. The following day, the cells were incubated for 48 hours in fresh medium or in medium containing 1 μM quercetin. These results shown are normalized to protein concentrations measured using the Bradford method and are representative of at least three independent experiments. The luciferase activity of each PRDX-Luc construct under normal conditions corresponds to 1. Bars represent the SD.
Figure 2.
 
(A) Schematic representations of the PRDX luciferase constructs PRDX2-Luc, PRDX3-Luc, PRDX4-Luc, and PRDX5-Luc, with their CpG islands and transcription factor binding sites. (B) Transcriptional activity of the PRDX2-5 genes in response to quercetin treatment. The indicated reporter plasmids were transiently transfected into NTM5 cells. The following day, the cells were incubated for 48 hours in fresh medium or in medium containing 1 μM quercetin. These results shown are normalized to protein concentrations measured using the Bradford method and are representative of at least three independent experiments. The luciferase activity of each PRDX-Luc construct under normal conditions corresponds to 1. Bars represent the SD.
Luciferase Assay
Transient transfection and a luciferase assay were performed as described previously. 29,30 Briefly, 1 × 105 NTM5 cells per well were seeded onto 12-well plates. The following day, cells were cotransfected with the indicated amount of reporter plasmid and expression plasmid using reagent (Superfect; Qiagen, Valencia, CA). For the luciferase assay using siRNA, siRNA-pretransfected 1.5 × 105 NTM5 cells, described above, were transfected with the indicated amounts of reporter plasmid at intervals of 12 hours. Forty-eight hours after transfection of reporter plasmid, cells were lysed with reporter lysis buffer (Promega). For quercetin treatment, 36 hours after transfection cells were further incubated under normal conditions or in the presence of 1 μM quercetin for 6 hours. Luciferase activity was detected using a luciferase assay system (PicaGene; Toyo-Inki, Tokyo, Japan. The light intensity was measured using a luminometer (Luminescencer JNII RAB-2300; Atto, Tokyo, Japan). The results shown are normalized to the protein concentration measured using the Bradford method and are representative of at least three independent experiments. 
Cytotoxicity Analysis
The water-soluble tetrazolium salt (WST-8) assay was performed as described previously. 24 Briefly, 2.5 × 103 NTM5 cells per well, transfected with siRNA as described, were seeded onto 96-well plates. The following day, to induce oxidative stress, cells were incubated with the indicated concentration of H2O2 in serum-free medium for 40 minutes. Then the medium was changed to the normal culture medium. After 72 hours, surviving cells were stained (TetraColor One; Seikagaku Corporation, Tokyo, Japan) for 90 minutes at 37°C. Absorbance was then measured at 450 nm. 
Statistical Analysis
Pearson correlation was used for statistical analysis, and significance was set at the 5% level. 
Results
Quercetin Induces PRDX Expression in TM Cells
We have previously shown that the PRDX1 gene is not expressed in immortalized human TM NTM5 cells. 24 To examine whether quercetin can activate PRDX family gene expression, NTM5 cells were treated with quercetin. As shown in Figure 1A, both PRDX3 and PRDX5 were induced by 1 μM quercetin in a time-dependent manner. We also found that both PRDX3 and PRDX5 were induced by quercetin treatment in a dose-dependent manner (Fig. 1B). We next investigated the effects of quercetin on the expression of the peroxiredoxin family in primary HTMCs. Although PRDX1 expression could not detected in immortalized TM cells, 24 HTMCs express PRDX1. 31 We again observed that the expression of both PRDX3 and PRDX5 was induced by the treatment of HTMCs with quercetin (Fig. 1C). 
Quercetin Enhances the Promoter Activity of Both the PRDX3 and PRDX5 Genes
We next investigated whether quercetin can activate the promoter activity of the PRDX genes using luciferase reporter assays. A schematic representation of the PRDX gene promoter is shown in Figure 2A. 23 Promoter activity of both the PRDX3 and PRDX5 genes was significantly enhanced approximately twofold to threefold by the quercetin treatment (Fig. 2B). We did a careful survey of the nucleotide sequences of the promoter of four PRDX genes and found several transcription factor binding sites, as shown in Figure 2A. The NRF1 binding sites are found in the promoters of both PRDX3 and PRDX5 but not in those of PRDX2 and PRDX4. 
Quercetin Induces Expression of the Transcription Factor NRF1
Next, we examined whether quercetin can induce the expression of a transcription factor that regulates PRDX gene expression. We initially investigated the cellular expression of NRF1 and found that the expression of NRF1 is localized primarily in nuclei. As shown in Figure 3, nuclear NRF1 was markedly increased after quercetin treatment in a time- and concentration-dependent manner. In contrast, there was no increase in the expression of the three transcription factors Ets1, Ets2, and Foxo3a. 
Figure 3.
 
(A) Quercetin treatment increases NRF1 but not Ets1, Ets2, or Foxo3a expression. NTM5 cells were incubated with 1 μM quercetin for the times indicated. Whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown under each blot. (B) NTM5 cells were for 12 hours cultured in the control medium or in medium containing the indicated concentrations of quercetin. Whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue.
Figure 3.
 
(A) Quercetin treatment increases NRF1 but not Ets1, Ets2, or Foxo3a expression. NTM5 cells were incubated with 1 μM quercetin for the times indicated. Whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown under each blot. (B) NTM5 cells were for 12 hours cultured in the control medium or in medium containing the indicated concentrations of quercetin. Whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue.
NRF1 Regulates the Expression of Both PRDX3 and PRDX5
To confirm the NRF1-dependent expression of both PRDX3 and PRDX5, we used specific siRNA for NRF1. Two independent siRNAs (#1 and #2) for NRF1 could effectively downregulate NRF1 expression. As shown in Figure 4A, protein expression of both PRDX3 and PRDX5 was significantly reduced by the transfection of two siRNAs for NRF1. Furthermore, we performed cotransfection experiments using the NRF1 expression plasmid with PRDX reporter plasmids. The reporter assays showed that NRF1 transactivated the promoter activity of both the PRDX3 and PRDX5 genes (Fig. 4B). On the other hand, the promoter activity of both the PRDX3 and PRDX5 genes was significantly downregulated by NRF1-specific siRNA transfection (Fig. 4C). 
Figure 4.
 
(A) NRF1 regulates PRDX3 and PRDX5 gene expression. Control siRNA (100 pmol) or NRF1#1 and #2 siRNA (100 pmol) were transfected into NTM5 cells. Whole nuclear lysates (100 μg for NRF1) and whole cell lysates (50 μg for PRDXs) were subjected to SDS-PAGE. The transferred membrane was blotted with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue. (B) NRF1 transactivates the promoter activity of the PRDX3 and PRDX5 genes. The indicated amount of NRF1 expression plasmid was transiently cotransfected with the indicated reporter plasmids into NTM5 cells. pGL3-P.V indicates the pGL3 promoter vector in which the luciferase gene is driven by the SV-40 promoter. Results were normalized to protein concentrations measured using the Bradford method. All values are representative of at least three independent experiments. The luciferase activity of each PRDX-Luc construct with transfection of the Flag vector corresponds to 1. Bars represent the SD. (C) Knockdown of NRF1 downregulates the promoter activity of the PRDX3 and PRDX5 genes. NTM5 cells were transiently transfected with the indicated amounts of control siRNA or NRF1 siRNA, followed by transfection with 0.5 μg of the indicated reporter plasmids at intervals of 12 hours. The results shown are normalized to protein concentrations measured using the Bradford method and are representative of at least three independent experiments. The luciferase activity of each PRDX-Luc construct with transfection of control siRNA corresponds to 1. Bars represent the SD.
Figure 4.
 
(A) NRF1 regulates PRDX3 and PRDX5 gene expression. Control siRNA (100 pmol) or NRF1#1 and #2 siRNA (100 pmol) were transfected into NTM5 cells. Whole nuclear lysates (100 μg for NRF1) and whole cell lysates (50 μg for PRDXs) were subjected to SDS-PAGE. The transferred membrane was blotted with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue. (B) NRF1 transactivates the promoter activity of the PRDX3 and PRDX5 genes. The indicated amount of NRF1 expression plasmid was transiently cotransfected with the indicated reporter plasmids into NTM5 cells. pGL3-P.V indicates the pGL3 promoter vector in which the luciferase gene is driven by the SV-40 promoter. Results were normalized to protein concentrations measured using the Bradford method. All values are representative of at least three independent experiments. The luciferase activity of each PRDX-Luc construct with transfection of the Flag vector corresponds to 1. Bars represent the SD. (C) Knockdown of NRF1 downregulates the promoter activity of the PRDX3 and PRDX5 genes. NTM5 cells were transiently transfected with the indicated amounts of control siRNA or NRF1 siRNA, followed by transfection with 0.5 μg of the indicated reporter plasmids at intervals of 12 hours. The results shown are normalized to protein concentrations measured using the Bradford method and are representative of at least three independent experiments. The luciferase activity of each PRDX-Luc construct with transfection of control siRNA corresponds to 1. Bars represent the SD.
Quercetin Induces NRF1 Expression through Nrf2 Activation
It has been shown that Nrf2 regulates NRF1 expression. 32 Nrf2 is a cytoplasmic protein translocated to the nuclei by oxidative stress. 33 We therefore examined whether quercetin can increase nuclear Nrf2 expression. As expected, quercetin did induce Nrf2 expression in both a time- and dose-dependent manner (Fig. 5A). Furthermore, we investigated whether the Nrf2 transcription factor can regulate the expression of NRF1 under quercetin treatment. We confirmed that quercetin-dependent induction of NRF1 was completely abolished by the transfection of Nrf2-specific siRNA (Fig. 5B). As a control, the expression of Foxo3a and PCNA was not affected by the transfection of siRNA under quercetin treatment. To examine the protective efficacy of quercetin treatment, TM cells were exposed to H2O2 in the presence of quercetin. Quercetin treatment significantly protected the TM cells against the cytotoxic activity of H2O2 (Fig. 5C). 
Figure 5.
 
(A) Effect of quercetin on expression of Nrf2. Left: NTM5 cells were incubated with 1 μM quercetin for the times indicated. Whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown under each blot. Right: NTM5 cells were cultured for 12 hours in the control medium or in medium containing the indicated concentrations of quercetin. Whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown under each blot. (B) NTM5 cells were transiently transfected with 100 pmol control or Nrf2 siRNAs. The following day, NTM5 cells were cultured in the control medium or in medium containing 1 μM quercetin. After 48 hours, whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed using the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown at the bottom of the panel. (C) Quercetin treatment counteracts H2O2 sensitivity in TM cells. Approximately 2.5 × 103 NTM5 cells were cultured in the control medium or in medium containing the indicated concentration of quercetin. The following day, to induce oxidative stress, cells were incubated with the indicated concentration of H2O2 in serum-free medium for 40 minutes After 48 hours, cell survival was analyzed using a WST-8 assay. All values are the means of at least three independent experiments. Bars represent the SD. CBB, Coomassie brilliant blue.
Figure 5.
 
(A) Effect of quercetin on expression of Nrf2. Left: NTM5 cells were incubated with 1 μM quercetin for the times indicated. Whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown under each blot. Right: NTM5 cells were cultured for 12 hours in the control medium or in medium containing the indicated concentrations of quercetin. Whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown under each blot. (B) NTM5 cells were transiently transfected with 100 pmol control or Nrf2 siRNAs. The following day, NTM5 cells were cultured in the control medium or in medium containing 1 μM quercetin. After 48 hours, whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed using the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown at the bottom of the panel. (C) Quercetin treatment counteracts H2O2 sensitivity in TM cells. Approximately 2.5 × 103 NTM5 cells were cultured in the control medium or in medium containing the indicated concentration of quercetin. The following day, to induce oxidative stress, cells were incubated with the indicated concentration of H2O2 in serum-free medium for 40 minutes After 48 hours, cell survival was analyzed using a WST-8 assay. All values are the means of at least three independent experiments. Bars represent the SD. CBB, Coomassie brilliant blue.
NRF1 Expression Modulates Cellular Sensitivity to H2O2
PRDX3 localizes to mitochondria and may protect mitochondrial DNA from ROS. NRF1 expression leads to the activation of genes concerned with mitochondrial biogenesis and protects cells from apoptosis. 34 As shown in Figure 6A, the downregulation of PRDX3 and PRDX5 sensitized TM cells approximately twofold against H2O2. On the other hand, the downregulation of NRF1 sensitized TM cells approximately fivefold against H2O2 (Fig. 6B). Among NRF1-regulated genes, mtTFA is important in protecting mitochondrial DNA from ROS-dependent apoptosis. 35 We showed that quercetin significantly induces mtTFA protein expression (Fig. 6C). 
Figure 6.
 
(A) Downregulation of PRDX3 and PRDX5 sensitizes TM cells to oxidative stress. Left: NTM5 cells were transiently transfected with 100 pmol control, PRDX2, or PRDX5 siRNA. After 72 hours, whole cell lysates (50 μg) were subjected to SDS-PAGE, and Western blot analysis was performed using the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown at the bottom of the panel. Right: approximately 2.5 × 103 NTM5 cells were transfected with 40 nM control siRNA (filled squares), PRDX3 siRNA (open rhomboids), or PRDX5 siRNA (open circles). Induction of the indicated concentrations of H2O2 was described for Figure 5C. All values are the means of at least three independent experiments. Bars represent the SD. (B) Downregulation of NRF1 sensitizes TM cells to oxidative stress. Approximately 2.5 × 103 NTM5 cells were transfected with 40 nM control siRNA (filled squares), NRF1#1 siRNA (open rhomboids), or NRF1#2 siRNA (open circles). Induction of the indicated concentrations of H2O2 was described for Figure 5C. All values are the means of at least three independent experiments. Bars represent the SD. (C) Effect of quercetin on the expression of mtTFA. Left: NTM5 cells were incubated with 1 μM quercetin for the times indicated. Whole cell lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. Right: NTM5 cells were cultured for 12 hours in the control medium or in medium containing the indicated concentration of quercetin. Whole cell lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue.
Figure 6.
 
(A) Downregulation of PRDX3 and PRDX5 sensitizes TM cells to oxidative stress. Left: NTM5 cells were transiently transfected with 100 pmol control, PRDX2, or PRDX5 siRNA. After 72 hours, whole cell lysates (50 μg) were subjected to SDS-PAGE, and Western blot analysis was performed using the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown at the bottom of the panel. Right: approximately 2.5 × 103 NTM5 cells were transfected with 40 nM control siRNA (filled squares), PRDX3 siRNA (open rhomboids), or PRDX5 siRNA (open circles). Induction of the indicated concentrations of H2O2 was described for Figure 5C. All values are the means of at least three independent experiments. Bars represent the SD. (B) Downregulation of NRF1 sensitizes TM cells to oxidative stress. Approximately 2.5 × 103 NTM5 cells were transfected with 40 nM control siRNA (filled squares), NRF1#1 siRNA (open rhomboids), or NRF1#2 siRNA (open circles). Induction of the indicated concentrations of H2O2 was described for Figure 5C. All values are the means of at least three independent experiments. Bars represent the SD. (C) Effect of quercetin on the expression of mtTFA. Left: NTM5 cells were incubated with 1 μM quercetin for the times indicated. Whole cell lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. Right: NTM5 cells were cultured for 12 hours in the control medium or in medium containing the indicated concentration of quercetin. Whole cell lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue.
Discussion
Quercetin has a wide variety of pharmacologic properties. 36 40 However, little is known about the mechanisms by which quercetin protects cells against oxidative stress. In the present study, we demonstrated that quercetin stimulates the antioxidant system through the activation of the Nrf2/NRF1 transcription pathway (Figs. 1 2 34). Quercetin is also a potent free radical scavenger, suggesting that quercetin would be an effective agent against oxidative stress-induced ocular diseases, including glaucoma. 
Several transcription factors are activated under oxidative stress induced by hydrogen peroxide and inflammatory cytokines, such as TNF-α and IL-1β. Among them, both NF-κB and Nrf2 are well-known transcription factors related to oxidative stress. 33,41 PRDXs can eliminate hydrogen peroxide efficiently and can participate in many physiological processes such as signal transduction and apoptosis. 42 There are six distinct members located in various subcellular compartments. PRDX1, PRDX2, and PRDX6 are in the cytoplasm, and PRDX3 is found in mitochondria. PRDX4 is in endoplasmic reticulum and is secreted. PRDX5 is found in various compartments. As shown in Figure 1C, the expression of five PRDXs was observed, and both PRDX3 and PRDX5 were induced by the treatment with quercetin in primary TM cells. We have previously shown that PRDX1 is not found in immortalized TM cells. 24 This might be due to the epigenetic mechanism because cellular transformation often induces epigenetic changes such as DNA methylation. 43 We have previously shown that oxidative stress induces PRDX1 and PRDX5 through the activation of Ets1. 23 Furthermore, PRDX2 expression is regulated by the transcription factor Foxo3a. 24 In this study, we found that the Nrf2/NRF1 transcription pathway is also involved in the expression of both the PRDX3 and PRDX5 genes (Figs. 4, 5). Nrf2, a basic leucine zipper transcription factor, is essential for the inducible and constitutive expression of several phase 2 detoxification proteins, including those required for mitochondrial respiratory function. 44,45 NRF1 was found to act on many nuclear genes required for mitochondrial respiratory function. 46 This primary function was confirmed by disrupting the Nrf1 gene in mice, resulting in a phenotype of peri-implantation lethality and a striking decrease in the mitochondrial DNA content of Nrf1-null blastocysts. 34  
One specific ROS, hydrogen peroxide, is produced by mitochondria. Because PRDXs can eliminate hydrogen peroxide efficiently, mitochondrial PRDX3 may protect mitochondrial DNA from ROS. 47 50 We have previously reported that a member of the high-mobility group protein family mtTFA can recognize oxidatively damaged DNA. 51,52 Furthermore, it has been shown that mtTFA binds to mitochondrial DNA (mtDNA) in the same way that histones bind to nuclear DNA. 53,54 Because mtTFA is not wrapped by chromatin proteins such as histones, it is highly sensitive to oxidative stress. mtTFA may protect mtDNA, acting as a guardian of mitochondrial function. 55,56 As shown in Figure 6C, quercetin induced mtTFA protein expression. This indicates that quercetin may protect mitochondria from oxidative stress through the induction of both mtTFA and PRDX3. We also demonstrated the protective activity of quercetin against H2O2 toxicity (Fig. 5C). Quercetin inhibits the activation of caspase 3 and abolishes the H2O2-dependent induction of apoptogenic proteins such as Bcl2. 57 This also suggests that quercetin inhibits the mitochondrial apoptotic pathway induced by various stresses. 
The endothelium plays a key role in the maintenance of anterior chamber homeostasis and also is involved in glaucoma pathogenesis. 58 Expression of the PRDX family was investigated in Fuchs' endothelial dystrophy, and the expression of PRDX2, PRDX3, and PRDX5 was significantly downregulated in this disease. 59 It has been reported that PRDX3 oxidation is found in TNF-α–treated cells and is the early event in apoptosis. This leads to an increase of hydrogen peroxide to modulate the progression of apoptosis. 60 These data indicate that the expression of PRDXs in endothelial cells may be also related to glaucoma pathogenesis. PRDX6 reduces oxidative stress– and TGF-β–induced abnormalities of TM cells. 61 TGF-β is a fibrogenic cytokine and increases ROS production, 62 indicating that our study may be relevant to the physiology and pathophysiology of the outflow pathway in glaucoma. 
To our knowledge, this is the first study showing modulation of an oxidative stress-protective pathway involving the control of PRDX3, PRDX5, and mtTFA expression by the transcription factors Nrf2 and NRF1. 
Footnotes
 Supported in part by Grants-in-Aid for Scientific Research from the Ministry for Education, Culture, Sports, Science and Technology of Japan (17016075), UOEH Grant for Advanced Research, and The Vehicle Racing Commemorative Foundation.
Footnotes
 Disclosure: N. Miyamoto, None; H. Izumi, None; R. Miyamoto, None; H. Kondo, None; A. Tawara, None; Y. Sasaguri, None; K. Kohno, None
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Figure 1.
 
(A) Effect of quercetin on PRDX expression. NTM5 cells were incubated with 1 μM quercetin for the times indicated. Whole cell lysates (50 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. (B) NTM5 cells were cultured for 12 hours in the control medium or medium containing the indicated concentrations of quercetin. Whole cell lysates (50 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. (C) Primary HTMCs were incubated with 1 μM quercetin for the times indicated. Whole cell lysates (50 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue.
Figure 1.
 
(A) Effect of quercetin on PRDX expression. NTM5 cells were incubated with 1 μM quercetin for the times indicated. Whole cell lysates (50 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. (B) NTM5 cells were cultured for 12 hours in the control medium or medium containing the indicated concentrations of quercetin. Whole cell lysates (50 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. (C) Primary HTMCs were incubated with 1 μM quercetin for the times indicated. Whole cell lysates (50 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue.
Figure 2.
 
(A) Schematic representations of the PRDX luciferase constructs PRDX2-Luc, PRDX3-Luc, PRDX4-Luc, and PRDX5-Luc, with their CpG islands and transcription factor binding sites. (B) Transcriptional activity of the PRDX2-5 genes in response to quercetin treatment. The indicated reporter plasmids were transiently transfected into NTM5 cells. The following day, the cells were incubated for 48 hours in fresh medium or in medium containing 1 μM quercetin. These results shown are normalized to protein concentrations measured using the Bradford method and are representative of at least three independent experiments. The luciferase activity of each PRDX-Luc construct under normal conditions corresponds to 1. Bars represent the SD.
Figure 2.
 
(A) Schematic representations of the PRDX luciferase constructs PRDX2-Luc, PRDX3-Luc, PRDX4-Luc, and PRDX5-Luc, with their CpG islands and transcription factor binding sites. (B) Transcriptional activity of the PRDX2-5 genes in response to quercetin treatment. The indicated reporter plasmids were transiently transfected into NTM5 cells. The following day, the cells were incubated for 48 hours in fresh medium or in medium containing 1 μM quercetin. These results shown are normalized to protein concentrations measured using the Bradford method and are representative of at least three independent experiments. The luciferase activity of each PRDX-Luc construct under normal conditions corresponds to 1. Bars represent the SD.
Figure 3.
 
(A) Quercetin treatment increases NRF1 but not Ets1, Ets2, or Foxo3a expression. NTM5 cells were incubated with 1 μM quercetin for the times indicated. Whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown under each blot. (B) NTM5 cells were for 12 hours cultured in the control medium or in medium containing the indicated concentrations of quercetin. Whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue.
Figure 3.
 
(A) Quercetin treatment increases NRF1 but not Ets1, Ets2, or Foxo3a expression. NTM5 cells were incubated with 1 μM quercetin for the times indicated. Whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown under each blot. (B) NTM5 cells were for 12 hours cultured in the control medium or in medium containing the indicated concentrations of quercetin. Whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue.
Figure 4.
 
(A) NRF1 regulates PRDX3 and PRDX5 gene expression. Control siRNA (100 pmol) or NRF1#1 and #2 siRNA (100 pmol) were transfected into NTM5 cells. Whole nuclear lysates (100 μg for NRF1) and whole cell lysates (50 μg for PRDXs) were subjected to SDS-PAGE. The transferred membrane was blotted with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue. (B) NRF1 transactivates the promoter activity of the PRDX3 and PRDX5 genes. The indicated amount of NRF1 expression plasmid was transiently cotransfected with the indicated reporter plasmids into NTM5 cells. pGL3-P.V indicates the pGL3 promoter vector in which the luciferase gene is driven by the SV-40 promoter. Results were normalized to protein concentrations measured using the Bradford method. All values are representative of at least three independent experiments. The luciferase activity of each PRDX-Luc construct with transfection of the Flag vector corresponds to 1. Bars represent the SD. (C) Knockdown of NRF1 downregulates the promoter activity of the PRDX3 and PRDX5 genes. NTM5 cells were transiently transfected with the indicated amounts of control siRNA or NRF1 siRNA, followed by transfection with 0.5 μg of the indicated reporter plasmids at intervals of 12 hours. The results shown are normalized to protein concentrations measured using the Bradford method and are representative of at least three independent experiments. The luciferase activity of each PRDX-Luc construct with transfection of control siRNA corresponds to 1. Bars represent the SD.
Figure 4.
 
(A) NRF1 regulates PRDX3 and PRDX5 gene expression. Control siRNA (100 pmol) or NRF1#1 and #2 siRNA (100 pmol) were transfected into NTM5 cells. Whole nuclear lysates (100 μg for NRF1) and whole cell lysates (50 μg for PRDXs) were subjected to SDS-PAGE. The transferred membrane was blotted with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue. (B) NRF1 transactivates the promoter activity of the PRDX3 and PRDX5 genes. The indicated amount of NRF1 expression plasmid was transiently cotransfected with the indicated reporter plasmids into NTM5 cells. pGL3-P.V indicates the pGL3 promoter vector in which the luciferase gene is driven by the SV-40 promoter. Results were normalized to protein concentrations measured using the Bradford method. All values are representative of at least three independent experiments. The luciferase activity of each PRDX-Luc construct with transfection of the Flag vector corresponds to 1. Bars represent the SD. (C) Knockdown of NRF1 downregulates the promoter activity of the PRDX3 and PRDX5 genes. NTM5 cells were transiently transfected with the indicated amounts of control siRNA or NRF1 siRNA, followed by transfection with 0.5 μg of the indicated reporter plasmids at intervals of 12 hours. The results shown are normalized to protein concentrations measured using the Bradford method and are representative of at least three independent experiments. The luciferase activity of each PRDX-Luc construct with transfection of control siRNA corresponds to 1. Bars represent the SD.
Figure 5.
 
(A) Effect of quercetin on expression of Nrf2. Left: NTM5 cells were incubated with 1 μM quercetin for the times indicated. Whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown under each blot. Right: NTM5 cells were cultured for 12 hours in the control medium or in medium containing the indicated concentrations of quercetin. Whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown under each blot. (B) NTM5 cells were transiently transfected with 100 pmol control or Nrf2 siRNAs. The following day, NTM5 cells were cultured in the control medium or in medium containing 1 μM quercetin. After 48 hours, whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed using the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown at the bottom of the panel. (C) Quercetin treatment counteracts H2O2 sensitivity in TM cells. Approximately 2.5 × 103 NTM5 cells were cultured in the control medium or in medium containing the indicated concentration of quercetin. The following day, to induce oxidative stress, cells were incubated with the indicated concentration of H2O2 in serum-free medium for 40 minutes After 48 hours, cell survival was analyzed using a WST-8 assay. All values are the means of at least three independent experiments. Bars represent the SD. CBB, Coomassie brilliant blue.
Figure 5.
 
(A) Effect of quercetin on expression of Nrf2. Left: NTM5 cells were incubated with 1 μM quercetin for the times indicated. Whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown under each blot. Right: NTM5 cells were cultured for 12 hours in the control medium or in medium containing the indicated concentrations of quercetin. Whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown under each blot. (B) NTM5 cells were transiently transfected with 100 pmol control or Nrf2 siRNAs. The following day, NTM5 cells were cultured in the control medium or in medium containing 1 μM quercetin. After 48 hours, whole nuclear lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed using the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown at the bottom of the panel. (C) Quercetin treatment counteracts H2O2 sensitivity in TM cells. Approximately 2.5 × 103 NTM5 cells were cultured in the control medium or in medium containing the indicated concentration of quercetin. The following day, to induce oxidative stress, cells were incubated with the indicated concentration of H2O2 in serum-free medium for 40 minutes After 48 hours, cell survival was analyzed using a WST-8 assay. All values are the means of at least three independent experiments. Bars represent the SD. CBB, Coomassie brilliant blue.
Figure 6.
 
(A) Downregulation of PRDX3 and PRDX5 sensitizes TM cells to oxidative stress. Left: NTM5 cells were transiently transfected with 100 pmol control, PRDX2, or PRDX5 siRNA. After 72 hours, whole cell lysates (50 μg) were subjected to SDS-PAGE, and Western blot analysis was performed using the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown at the bottom of the panel. Right: approximately 2.5 × 103 NTM5 cells were transfected with 40 nM control siRNA (filled squares), PRDX3 siRNA (open rhomboids), or PRDX5 siRNA (open circles). Induction of the indicated concentrations of H2O2 was described for Figure 5C. All values are the means of at least three independent experiments. Bars represent the SD. (B) Downregulation of NRF1 sensitizes TM cells to oxidative stress. Approximately 2.5 × 103 NTM5 cells were transfected with 40 nM control siRNA (filled squares), NRF1#1 siRNA (open rhomboids), or NRF1#2 siRNA (open circles). Induction of the indicated concentrations of H2O2 was described for Figure 5C. All values are the means of at least three independent experiments. Bars represent the SD. (C) Effect of quercetin on the expression of mtTFA. Left: NTM5 cells were incubated with 1 μM quercetin for the times indicated. Whole cell lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. Right: NTM5 cells were cultured for 12 hours in the control medium or in medium containing the indicated concentration of quercetin. Whole cell lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue.
Figure 6.
 
(A) Downregulation of PRDX3 and PRDX5 sensitizes TM cells to oxidative stress. Left: NTM5 cells were transiently transfected with 100 pmol control, PRDX2, or PRDX5 siRNA. After 72 hours, whole cell lysates (50 μg) were subjected to SDS-PAGE, and Western blot analysis was performed using the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown at the bottom of the panel. Right: approximately 2.5 × 103 NTM5 cells were transfected with 40 nM control siRNA (filled squares), PRDX3 siRNA (open rhomboids), or PRDX5 siRNA (open circles). Induction of the indicated concentrations of H2O2 was described for Figure 5C. All values are the means of at least three independent experiments. Bars represent the SD. (B) Downregulation of NRF1 sensitizes TM cells to oxidative stress. Approximately 2.5 × 103 NTM5 cells were transfected with 40 nM control siRNA (filled squares), NRF1#1 siRNA (open rhomboids), or NRF1#2 siRNA (open circles). Induction of the indicated concentrations of H2O2 was described for Figure 5C. All values are the means of at least three independent experiments. Bars represent the SD. (C) Effect of quercetin on the expression of mtTFA. Left: NTM5 cells were incubated with 1 μM quercetin for the times indicated. Whole cell lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. Right: NTM5 cells were cultured for 12 hours in the control medium or in medium containing the indicated concentration of quercetin. Whole cell lysates (100 μg) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of β-actin is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue.
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