Fig. 4.

Endogenous sCLU localizes in the mitochondria and the nucleocytosolic continuum of U-2 OS cells. Immunoblot analyses of isolated cytosolic (cyt; A), mitochondrial (mt; A), and cytoplasmic and nuclear fractions (C) probed with an anti-sCLU antibody. Blots in C were also probed with antibodies against p53, p21, Bax, Bcl-2, and Ku70. Fraction purity in A was verified by anti-OxPhos complex IV (OxP-IV) probing (mitochondrial marker; shown in Fig. 5A), and in C by blot probing with anti-proliferating cell nuclear antigen (exclusively cytosolic in osteosarcoma cells) and anti-lamin A/C (nuclear marker). B, CLSM immunofluorescence (top and bottom) or TEM immunogold (middle) localization of sCLU protein in control and sCLU knocked-down cells. For CLSM immunolocalization, cells were costained with an anti-sCLU (green), MitoTracker (mitochondria specific stain; red), and 4’,6-diamidino-2-phenylindole dihydrochloride (blue). Captured images were merged to reveal codistribution sites (yellow for green-red and cyan for green-blue). Top, arrows, mitochondria; arrowheads, nuclei. Bottom, dashed arrow, aggregated mitochondria in sCLU-depleted cells. Middle, sCLU-related gold particles after immunogold TEM localization were found in mitochondria (mt; see also inset), cytosol (cyt), and nucleus (nuc). Bar, 10 µm (CLSM) and 100 nm (TEM). D, immunoblotting of nonextractable pelleted material and the soluble cytosol after hypotonic lysis of control cells. Blots were probed with antibodies against sCLU, OxPhos complex IV, Bcl-2 (membrane bound), Ku70 (nuclear and cytosolic distribution), and proliferating cell nuclear antigen. All assays were done 72 h post-siRNA transfection.