Figure 3. Western blot analysis of fibronectin and α-SMA in HLE-B3 cells treated with SB216763 +/− hypoxia inducible factor translation inhibitors. HLE-B3 cells were cultured in 25 cm² flasks with 20% fetal bovine serum (FBS) and switched to serum-free media for 24 h before the experiment. The cells were incubated with 3 ml of serum-free media containing 12 µm SB216763 or SB216763 combined with either 0.5 µm hypoxia-inducible factor-1α (HIF-1α) translation inhibitor (KC7F2) or HIF-2α translation inhibitor (CAS882268–69–1). Total cell lysates were collected from the HLE-B3 cell cultures. A portion of the sample was used for protein quantification using the EZQ Protein Quantification Kit and 3X sodium dodecyl sulfate (SDS) buffer was added to the remaining lysates, which were subsequently boiled for 5 min; the proteins were resolved by electrophoresis on 12% SDS-polyacrylamide gels (20 μg protein/lane). Proteins were then transferred to nitrocellulose membranes. The experiment was repeated twice with independent cell cultures. A and C show the lysate from one cell sample, while B and D show the lysate from the second, independent cell sample. The normalized lysates were analyzed for alpha smooth muscle actin (α-SMA) and fibronectin using ImageJ analysis. There was a significant increase in the in the SB216763-treated samples compared to the corresponding expression of both epithelial to mesenchymal transition (EMT) marker proteins, fibronectin and α-SMA compared to corresponding control samples treated with dimethyl sulfoxide (DMSO) (p<0.05). SB216763-treated samples with added HIF-2α translation inhibitor showed similar results. The asterisk (*) signifies there was a statistically significant increase in the levels of fibronectin and α-SMA in the SB only treated and SB+HIF-2α treated samples relative to the control (DMSO) sample (p<0.05). In marked contrast, the SB216763-treated samples with the added HIF-1α translation inhibitor revealed substantially suppressed expression of α-SMA and fibronectin. SB=SB216763.