Fig 3. Effects of SHP or GW4064 on HIF-1α expression.

(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, p ≤ 0.1; b, p ≤ 0.05; c, p ≤ 0.01; d, p ≤ 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.