FIG. 2.
Detection of mitochondrial O2−• generation stimulated by HF-LPLI (200 mW/cm2 at 10 min). (A) Monitoring of initial ROS generation stimulated by HF-LPLI. The fluorescence images of A549 cells overlaid with H2DCFDA, SE dye, and DsRed-mit fluorescence protein were acquired immediately after PDT or HF-LPLI treatment. (B) Monitoring of initial O2−• generation stimulated by HF-LPLI. The fluorescence images of ASTC-a-1 and A549 cells overlaid with DHE and MTR dyes were acquired immediately after HF-LPLI treatment. (C) Subcellular fractions (MITO, mitochondria; CYTO, cytosol; NUCL, nucleus; PLAS, plasma membrane) of primary mouse liver cells pre-incubated with FCLA were subjected to HF-LPLI. Data were collected during irradiation. Subcellular fractions were pre-cultured with SOD (50 U/ml) 1 h before HF-LPLI. (D) The dynamics of mitochondrial O2−• generation. The sequential images of ASTC-a-1 cells stained with MitoSOX™ dye were acquired after PDT or HF-LPLI treatment. (E) The quantitative analysis of the mitochondrial O2−• generation is shown in (D). The data represent the mean±SD (n=5). (F) Ratio of GSH and GSSG in ASTC-a-1 and A549 cells 2 h after HF-LPLI treatment. The data represent the mean±SD (n=5). *P<0.05 versus control. (G) Western blot analysis of MnSOD levels in protein extracts from ASTC-a-1 and A549 cells 2 h after HF-LPLI. β-Actin was blotted as a control for protein loading. DHE, dihydroethidium; FCLA, fluoresceinyl cypridina luciferin analog; GSH, glutathione; GSSG, oxidized glutathione; MnSOD, manganese superoxide dismutase; MTR, MitoTraker Deeper Red 633; O2−•, superoxide anion; SOD, superoxidase dismutase. To see this illustration in color, the reader is referred to the web version of this article at www. liebertpub.com/ars