Angiostatin binding activity and colocalization with ATP synthase in mitochondria. (A) Endothelial cell lines HUVECs and HMVECs and tumor cell lines AsPC-1 and A2058 were subjected to FACS analysis using angiostatin concentrations of 1 (blue) and 10 μg/mL (green). The red area represents the control Fc binding. (B) IF analysis of HUVEC and A2058 under permeable (P) and nonpermeable (NP) conditions. No staining was observed with control Fc. (C) HUVECs and (D) A2058 cells were fixed under permeabilized conditions and incubated with hFcAS (50 ng/mL) for 2 hours and detected by AlexaFluor 488 anti–human IgG (green). Those cells were also double stained by anti-ATP synthase β subunit antibody, mitochondria marker (MitoTracker), or actin staining phalloidin and detected by Alexa Fluor 594 antibody (red). In merged (last column), hFcAS and ATP synthase β subunit or MitoTracker showed colocalization (yellow) in both endothelial and tumor cells by confocal microscopy. DAPI staining of nuclei is shown in blue. Bar represents 20 μm.