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 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 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