Fig. 5

Download : Download high-res image (219KB)Download : Download full-size image Figure 5. LDL-derived fatty acids regulate HIFα. A, immunoblots (WB) of whole-cell lysates from HIF reporter cells cultured for 24 h in FBS with the indicated concentrations of lalistat 2, a specific LAL inhibitor. LAMIN A/C served as a loading control. B, flow cytometry analysis from HIF reporter cells cultured for 24 h in FBS, LPDS, or FBS with lalistat (25 μm) or U18666A (2 μm). C, Pa03c cells were cultured for 16 h in FBS, LPDS, or FBS with lalistat (25 μm) or U18666A (2 μm). Gene expression for SREBP or HIF transcriptional targets measured by RT-qPCR was normalized to vehicle-treated Pa03c cells cultured in FBS. Error bars, S.E. of -fold changes from three biological replicates (mean ± S.E.). D, PHD activity assay of cell lysates from Pa03c cells cultured in FBS, LPDS, or FBS with lalistat (25 μm) or U18666A (2 μm) for 16 h. Four-parameter log logistic models were fit to data obtained from at least three independent experiments. The bar plot shows the calculated PHD activities from the curves at 25 µg. ***, p < 0.0005, Student's t test. E, flow cytometry analysis from HIF reporter cells cultured for 24 h in FBS, LPDS, or LPDS supplemented with methyl-β-cyclodextrin complexed cholesterol (chol., 25 μm) or albumin-conjugated oleic acid (OA; 800 μm). F, immunoblots of nuclear extracts from WT Pa03c cells (n = 3) cultured for 16 h in FBS or LPDS in the absence or presence of water-soluble methyl-β-cyclodextrin-cholesterol complex (MβCD-chol) or in the presence of bovine lipoproteins (100 mg/dl). p values from a single-column t test (LPDS with MβCD-chol versus LPDS) are shown. ns, not significant; *, p < 0.05; **, p < 0.005. G, PHD activity assay of cell lysates from Pa03c cells cultured in FBS, in LPDS with fatty acid–free albumin (0.75%), or in fatty acid–free albumin–conjugated oleic acid (800 μm) for 16 h. Four-parameter log logistic models were fit to data obtained from at least three independent experiments. The bar plot shows the calculated PHD activities from the curves at 25 µg. ***, p < 0.0005; **, p < 0.005, Student's t test.