Fig. 6

Aberrant HIF1α signaling causes mitochondrial dysfunction. a, b Immunofluorescence detection of COX4 and TOM20 was performed in lumbar discs of WT and Vhl cKO mice (n = 3 per group). c, d Percentages of COX4- and TOM20-immunoreacted positive cells. e RCS cells were transfected with siRNA-Vhl (RCS-siRNA-Vhl). Total RNA was isolated, and the mRNA levels of Ppargc1a, Mxi1, Mfn1, Mfn2, Opa1, Ant1, CypD, and Ucp3 were detected by RT-PCR (n = 3 per group). f, g Fluorescent staining for monitoring mitochondria with a MitoTracker® probe. (h-i) RCS cells subjected to siRNA-Vhl were stained with JC-1. Changes in △Ψm were detected by fluorescence microscopy. (j, k) ROS production was determined using the fluorescent probe dihydroethidium (DHE) (green: DHE; blue: Hoechst) (n = 3–4). l Quantification of flow cytometry for mitochondrial mass. m Carboxymethyl lysine (CML) signals were analyzed by an immunofluorescence assay from WT and Vhl cKO mice at the age of 12 months (red: anti-CML; blue: Hoechst) (n = 3–4). n Quantification of mitochondrial membrane potential, o DHE fluorescence, p areas of CML-positive cells in EP, and q percentages of CML-positive cells in AF. Scale bar: 50 µm (a–b, m), 20 µm (h–k), 10 µm (f, g). Data are expressed as the percent expression relative to controls. Values represent the mean (symbols) ± SD (error bars). *P < 0.05; **P < 0.01; ***P < 0.001.