Figure 6. Effects of CO on Mitochondria in PCa cells.

A. OCR and ECAR were measured in PC3 cells treated with medium bubbled with 250 ppm CO. Relative mitochondria activity was measured over time and the levels are shown at 30 min. B. OCR was measured in PC3 or PNT1A ± Rotenone (20 nM) for 10 min followed by 30 min ± CO (250 ppm). Data are representative for 2 independent experiments in duplicates. C. The levels of glucose and lactate were measured in the lysates of PC3 cells ± CO (250ppm) for 6 hours. D–E. Mitochondrial superoxide production was measured by Flow Cytometry using MitoSox. Data are representative for 3 independent experiments in triplicates. **p<0.01. F. Cell cycle analysis was performed in PC3 and PNT1A cells by PI staining. Cells were synchronized for 48 h via serum-starvation and then treated for 48h with FBS-containing medium ± CO. Data are representative of 3 independent experiments in triplicates. **p<0.01. G. SPECT analysis in PC3 cells ± CO (250 ppm) or CORM-A1 (20 or 100 µM) for 24h. Intensity of radiation is presented as a mean ± SD of 2 independent experiments performed in triplicate. *p<0.05 vs control, **p<0.01 vs control. H. Immunohistochemical staining for cytochrome C in subcutaneous prostate cancer xenografts as an indicator of cell death. Representative sections from n=3–4 animals are shown. Scale bar, 150 µm. I–J. MitoTracker Red CMXRos staining of PC3 cells treated with Doxorubicin (Dox) ± CO for 4h. Hoechst was used to stain nuclei. Representative pictures are shown in C and densitometric intensity analyses of 3 fields is shown in D. Results represent mean ± SD of 3 independent experiments. *p<0.001 vs control. Scale bar, 10 µm. K. Immunoblotting with antibodies against mtTFA in the lysates of CO treated PC3 and A549 cells (4h, 250 ppm). L. Immunofluorescence staining with antibody against vimentin in lung adenocarcinoma cell line A549, control and Rhoº (EtBr) ± CO (250 ppm, 48h). Data are representative of 2 independent experiments in duplicate. Scale bar, 30 µm.