Fig. 4

Figure 4. LDL regulation of HIFα requires mitochondria. A, immunoblots (WB) of whole-cell lysates from HIF reporter cells cultured for 24 h in LPDS, LPDS with LDL (1 mg/ml), or LPDS with LDL (1 mg/ml) at 1% O2 with the indicated antioxidants: ascorbate (Ascor; 25 μm), ebselen (Eb; 25 μm), PDTC (20 μm), or 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS; 15 μm). LAMIN A/C served as a loading control. B, immunoblots of whole-cell lysates from HIF reporter cells cultured for 24 h in LPDS, LPDS with LDL (1 mg/ml), or LPDS with LDL (1 mg/ml) at 1% O2 with the indicated mitochondrial complex inhibitors (Complex-i): I (rotenone, 2 μm), II (malonate, 5 mm), III-A (antimycin, 10 μm), III-M (myxothiazol, 1 μm), or IV (oligomycin, 2 μm). LAMIN A/C serves as a loading control. C, immunoblots of whole-cell lysates from HIF reporter cells cultured for 24 h in LPDS with LDL (1 mg/ml); LPDS with mitochondrial-targeted chemical TPMP (1 μm) or antioxidant MitoQ (0.008–1 μm); or LPDS with LDL (1 mg/ml) plus the iron chelator (DFO; 100 μm) and mitochondrial-targeted antioxidant MitoQ (1 μm). LAMIN A/C served as a loading control. D, PHD activity assay of cell lysates from Pa03c cells cultured for 16 h in FBS, LPDS, or LPDS with mitochondrial-targeted chemical TPMP (1 μm) or antioxidant MitoQ (1 μm). 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, immunoblots of nuclear extracts from Pa03c cells or UQCRFS1 KO Pa03c cells cultured for 16 h in LPDS with LDL (1 mg/ml) or LPDS or FBS with DMOG (1 μm). LSD1 served as a loading control. F, succinate and fumarate levels in cell extracts measured by NMR from Pa03c cells cultured in FBS or LPDS for 16 h. NS, p > 0.05, Student's t test. Error bars, S.E.