Effect of CDCA on hypoxic target genes.

<p>(A) Experimental scheme. HepG2 cells were serum starved with medium containing 0.5% FBS for 20 hours prior to CDCA (100 μM or indicated dose) treatment. 6 hours after CDCA treatment, the cells were exposed to 20%, 5% or 0.1% O₂ for the indicated hours. (B) Quantitative RT-PCR analyses of SHP mRNA. The expression level was normalized with the expression level of 18s rRNA. (C) Western analyses for SHP and β-actin. β-actin protein was detected as a loading control. Data shown are representative of three experiments (C) Quantitative RT-PCR analyses of carbonic anhydrase 9 (CA9), phosphoglycerate kinase1 (PGK1), endoplasmic reticulum oxidoreductin 1-like (EROL1), lysyl oxidase (LOX), prolyl 4-hydroxylase, alpha peptide 1 (P4HA1). a, <i>p</i> ≤ 0.1; b, <i>p</i> ≤ 0.05; c, <i>p</i> ≤ 0.01; d, <i>p</i> ≤ 0.001; e, <i>p</i> = 0.197; f, <i>p</i> = 0.724.</p

Effect of CDCA on HIF-1α expression.

<p>After starvation for 20hr, HepG2 cells were pretreated with CDCA (100 μM or indicated dose) for 6 hours then exposed to 20%, 5% or 0.1% O₂ for the indicated hours. (A) Western analyses for HIF-1α, 14-3-3γ and β-actin proteins. 14-3-3γ and β-actin proteins were detected as loading controls. (B) Quantitative RT-PCR of HIF-1α mRNA. (C and D) Western analyses of HIF-1α protein. HepG2 cells which were serum starved with medium containing 0.5% FBS for 20 hours prior to stimulation with MG132 (10 μM) and/or CDCA. 6 hours after treatment, the cells were exposed to 20% or 5% O₂ for 4 hours. Ubiquitinated and original HIF-1α proteins are indicated. HDAC1 protein or β-actin protein were examined in order to verify equal loading. (D) 20 μg of MG132 untreated total cell extracts and 5 μg of MG132 treated total cell extracts are loaded, respectively.</p

Effects of SHP or GW4064 on HIF-1α expression.

<p>(A and B) HepG2 cells were transfected with an empty vector or pcDNA3/HA-SHP. 18 hours after transfection, the cells were serum starved with medium containing 0.5% FBS for 20 hours. The cells were treated DMSO or 100 μM of CDCA for 6 hours in the absence or presence of MG132 and then exposed to 20%, 5% or 0.1% O₂ for 4 hours. 30 μg of total cell extracts are loaded for western analyses. (C to E) After starvation for 20hr, HepG2 cells were pretreated with GW4064 (GW) (5 μM or indicated dose) for 6 hours then exposed to 20%, 5% or 0.1% O₂ for the indicated hours. (C and E) qRT-PCR analyses of SHP or HIF-1α respectively. The expression level was normalized with the expression level of 18s rRNA. a, <i>p</i> ≤ 0.1; b, <i>p</i> ≤ 0.05; c, <i>p</i> ≤ 0.01; d, <i>p</i> ≤ 0.001. (D) Western analyses of HIF-1α, SHP and β-actin. (F) Western analyses of HIF-1α and β-actin in HepG2 cells which were treated with indicated doses of GW4064 and MG132 as described above. Ubiquitinated and original HIF-1α proteins are indicated. β-actin protein were examined in order to verify equal loading.</p

Effect of CDCA on de novo synthesis of HIF-1α protein.

<p>(A) Experimental scheme. After serum starvation for 20 hr, HepG2 cells were pretreated with DMSO or CDCA (100μM), 30 min prior to MG132 (10 μM) treatment. (B) Western analyses of HIF-1α and β-actin proteins (three western blots are shown). (C) Experimental scheme. HepG2 cells were pretreated with CHX (10 μg/ml, 3 hr), then the culture media were replaced with fresh media containing MG132 (10 μM) and/or CDCA (100 μM) as indicated. (D) Western analyses of HIF-1α and β-actin proteins (three western blots are shown). Quantification of western analyses. The intensities of HIF-1α and β-actin bands marked with bars were measured using Image J software. The y-axis indicates the relative band intensities of HIF-1α protein to 0 hr. The band intensities of HIF-1α protein were normalized by β-actin protein. The x-axis indicates the hours for MG132 treatments. <i>p</i> values between band intensities of CDCA-treated and untreated samples are shown. a, <i>p</i> ≤ 0.1; b, <i>p</i> ≤ 0.05; c, <i>p</i> ≤ 0.01; d, <i>p</i> ≤ 0.001; e, <i>p</i> = 0.251.</p

Hypoxic activation of HIF-1α directly regulates the transcriptional activity of ERRγ.

<p>(<b>A</b>) HepG2 cells were transfected with hERRγ-Luc. After 24 h of the transfection, HepG2 cells were exposed to hypoxia for indicated time period. Experiments were carried out in triplicate and data are expressed as the fold activation relative to the control. (<b>B–C</b>) HepG2 cells were transfected with hERRγ (−2 kb)-Luc, hERRγ (−1 kb)-Luc, hERRγ (−0.5 kb)-Luc, hERRγ (−0.3 kb)-Luc, hERRγ (HREmt1)-Luc, hERRγ (HREmt2)-Luc, hERRγ (HREmt1+2)-Luc. After 24 h of the transfection, HepG2 cells were exposed to hypoxia for 9 hr and analyzed using luciferase and β-galactosidase assay. Experiments were performed in duplicate and data are expressed as the fold activation relative to the control. (<b>D</b>) ChIP assay: HepG2 cell was exposed to hypoxia for 9 hr. Input represents 10% of purified DNA in each sample. Cell extracts were immunoprecipitated with anti-HIF-1α and purified DNA samples were employed for Q-PCR with primers binding to HRE1 (−1080 to −849) and HRE2 (−508 to −295) and distal site (−1826 to −1586) on the <i>ERRγ</i> gene promoter. All data are representative of at least three independent experiments. Error bars show ± S.E.M. ^***<i>P</i><0.001 by two-tailed Student <i>t</i>-test.</p

ERRγ inverse agonist GSK5182 down-regulates the hypoxia-induced PDK4 expression.

<p>(<b>A–B</b>) HepG2 cells were transfected with hPDK4-Luc. After transfection, the cells were exposed in hypoxia and treated with or without GSK5182. Harvested lysates were utilized for luciferase and β-galactosidase assay (A). Q-PCR was performed using isolated total RNA (B). Experiments were done in triplicate and data are expressed as the fold activation relative to the control. (<b>C</b>) HepG2 cells were seeded in 60-cm² dishes and incubated overnight. The cells were incubated in hypoxia and treated with or without GSK5182. Total protein was harvesed for Western blot analysis using indicated antibodies. (<b>D</b>) HepG2 cells were seeded in 60-cm² dishes and incubated overnight. The cells were treated with or without chemicals (DFO and GSK5182) for 6 hr. Total protein and mRNA were isolated for Western blot assay and RT-PCR and normalized with α or β-tubulin and β-actin. (<b>E</b>) A schematic representation. All data are representative of at least three independent experiments. Error bars show ± S.E.M. ^**<i>P</i><0.01, ^***<i>P</i><0.001 by two-tailed Student <i>t</i>-test.</p

Hypoxia induces the ERRγ gene expression in hepatoma cell lines.

<p>(<b>A–B</b>), HepG2 cells were seeded in 60-cm² dishes and incubated overnight. Then cells were incubated under hypoxia at indicated time period. The expression of ERRγ was analyzed by Western blot (<b>A</b>) and Q-PCR (<b>B</b>) analysis. (<b>C–D</b>) HepG2 cells were seeded in 60-cm² dishes and incubated overnight and then treated with DFO at indicated concentration and time period. The expression of ERRs was analyzed by Western blot (<b>C</b>) and Q-PCR (<b>D</b>) analysis. ERRγ gene expression was normalized to L32 gene expression, and α or β-tubulin expression. All data are representative of at least three independent experiments. Error bars show ± S.E.M. ^*<i>P</i><0.05, ^**<i>P</i><0.01 by two-tailed Student <i>t</i>-test.</p

HIF-1α increases the expression of ERR γ.

<p>(<b>A</b>) HepG2 cells were transfected with nonspecific siRNA (NS) or si-HIF-1α, and isolated total protein was analyzed by Western blot. α-tubulin was used as a control. (<b>B</b>) HepG2 cells were transfected with pcDNA3-HIF-1α, pcDNA3-ARNT and hERRγ-Luc, respectively. Experiments were conducted in duplicate and data are expressed as the fold activation relative to the control. (<b>C</b>) HepG2 cells were transfected with pcDNA3-HIF-1α and pcDNA3-ARNT and Q-PCR was performed using isolated total RNA. (<b>D–E</b>) HepG2 cells were transfected with nonspecific siRNA (NS) or si-HIF-1α. After transfection, lysates were utilized for luciferase and β-galactosidase assay (D). Q-PCR was performed using isolated total RNA from HepG2 cells (E). All data are representative of at least three independent experiments. Error bars show ± S.E.M. ^*<i>P</i><0.05, ^***<i>P</i><0.001 by two-tailed Student <i>t</i>-test.</p

ERRγ directly regulates hypoxia mediated PDK4 gene expression.

<p>(<b>A</b>) HepG2 cells were transfected with hPDK4-Luc. After transfection, the cells were exposed to hypoxia for indicated period and lysates were utilized for luciferase and β-galactosidase assay. Experiments were done in triplicate and data are expressed as the fold activation relative to the control. (<b>B</b>) HepG2 cells were seeded in 60-cm² dishes and trasnfected siERRγ and control siRNA for 72 hr and then exposed hypoxia for 9 hr. Cells were harvested for analyzing luciferase and β-galactodidase assay. (<b>C–D</b>) HepG2 cells were transfected with several deletion constructs of hPDK4 (−848)-Luc, hPDK4 (−500)-Luc, hPDK4 (−291)-Luc and hPDK4-mtERRE1-Luc with pcDNA3-ERRγ in the presence or absence with hypoxia exposure, respectively. 48 hr after transfection, the cells were harvested and performed luciferase and β-galactodidase assay. Experiments were done in duplicate and data expressed as the fold activation related to control. (<b>E</b>) HepG2 cell were transiently transfected with hPDK4 (−848)-Luc, hPDK4-mtERRE1-Luc, pcDNA3-ERRγ and pcDNA3-HIF-1α. (<b>F</b>) ChIP assay: HepG2 cell was exposed to hypoxia for 9 hr. Input represents 10% of purified DNA in each sample. Cell extracts were immunoprecipitated with anti-ERRα and purified DNA samples were employed for Q-PCR with primers binding to ERRE (−502 to −252) and distal site (−1056 to −886) on the <i>PDK4</i> gene promoter. All data are representative of at least three independent experiments. Error bars show ± S.E.M. ^***<i>P</i><0.001 by two-tailed Student <i>t</i>-test.</p

ERRγ mediates the hypoxia induced expression of PDK4.

<p>(<b>A–B</b>) HepG2 cells were seeded in 60-cm² dishes and exposed to hypoxia for indicated time period. Total RNA and protein were isolated and used for Western blot (<b>A</b>) and Q-PCR (<b>B</b>), respectively. (<b>C–D</b>) HepG2 was treated with DFO for indicated concentration and time period and then cells were harvested. Total protein was harvested for Western blot (<b>C</b>) using indicated antibodies and was normalized to β-tubulin expression. And total RNA was isolated for Q-PCR (<b>D</b>). The mRNA levels of PDK2 and PDK4 were normalized to L32 gene expression. (<b>E</b>) Effect of knockdown of ERRγ. HepG2 cells were infected with Ad-US and Ad- shERRγ for 48 hr, respectively. Total protein was isolated for Western blot analysis of PDK4 and then was normalized to β-tubulin or α-tubulin expression. All data are representative of at least three independent experiments. Error bars show ± S.E.M. ^*<i>P</i><0.05 by two-tailed Student <i>t</i>-test.</p