Tcs sp2

Samples were prepared as described previously (Tenconi et al., 2013 (link)). Samples were examined under Leica TCS-SP2 and Leica TCS-SP5 confocal laser-scanning microscopes. SYTO9 and SYTOX stained samples were examined at a wavelength of 488 for excitation and 530 nm (green) for emission. Red autofluorescence of PdGs and propidium iodide-stained samples were examined at a wavelength of 543 nm (Leica TCS-SP2) or 568 nm (Leica TCS-SP5) for excitation and 630 nm (red) for emission as described previously (Tenconi et al., 2012 (link)). Quantitative analyses were performed employing the Leica LAS-AF image analysis program. Image processing and 3D reconstruction of Streptomyces filaments were performed as described previously (Tenconi et al., 2013 (link)).

Normal and tumor cells were seeded at a density of 2×10⁵ on glass coverslips and left to adhere overnight at 37°C in 5% CO2. In the first part of experiment, the cells were exposed to 250μM of 6-AN for 24h, or incubated in medium alone, as previously described. In the second part of experiment, the cells were exposed to 250μM of 6-AN for 24h and then were treated with 100 μM of ascorbic acid 2-phosphate (AA2P) for 24h. After several washes to remove AA2P the cells were incubated for 72h. To study mitochondrial superoxide generation, cells were stained with 5 μM MitoSOX red for 10 min at 37°C and washed three times before imaging. Cells were fixed using ice-cold 4% paraformaldehyde for 10 min at room temperature. Then, nuclei were revealed by counterstaining with TO-PRO-3 (Molecular Probes). Images were taken with a Leica TCS SP2 (Leica, Wetzlar, Germany) confocal laser-scanning microscope using an ×63 objective lens.

The tumor and control cells were fixed in 4% paraformaldehyde (PFA), rinsed with phosphate-buffered saline (PBS, cod. P4417, Sigma-Aldrich, St. Louis, MO, USA) and then exposed to primary antibodies diluted in PBS with 0.2% BSA (Table 3). Then, the cells were incubated for 45 min with the corresponding secondary antibodies at 37 °C (Table 3), and, following washing, they were incubated for 20 min with 0.01% TO-PRO-3 (Invitrogen, Carlsbad, CA, USA) for nuclear staining, and mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA). The cells were examined under a Leica TCS SP2 (Leica, Wetzlar, Germany) confocal laser scanning microscope using 40× and 63× objective lenses with either 1× or 2× zoom factors. A sequential scan procedure was applied during image acquisition of the two fluorophores. Confocal images were taken at 200 nm intervals through the z axis of the section. The images from individual optical planes and multiple serial optical sections were analyzed, digitally recorded, and stored as TIFF files using Adobe Photoshop software (Adobe Systems Inc. San Jose, CA, USA).

The characterization and localization of PTX3 signal were investigated on frozen tissue included in OCT medium (Tissuetek). The slides were incubated with 5% rabbit serum for 1 hour at 37° C. Slides were then incubated for 1 hour at room temperature with specific antibodies. After three washes in PBS, slides were then incubated with the appropriate secondary antibodies(Alexa Flour 488 and 555, Molecular Probes, Eugene, OR). All sections were counterstained with TO-PRO-3 (Molecular Probes). Negative controls were prepared with irrelevant antibodies. The sections were analyzed using the Leica TCS SP2 (Leica, Wetzlar, Germany) confocal laser-scanning microscope. The number of infiltrating cells was measured in at least10 high power (x630) fields/section by two independent observers blinded to the origin of the slides. The final counts were the mean of the two measures. In no case interobserver variability was higher than 20%.

For the cell proliferation assay, 2 × 10⁵ Caki-2 cells were plated in a 10 cm dish, 1 day before exposure to siRNA NDUFA4L2 or to medium alone, and cultured in normoxic conditions. The cells were exposed to 50nM of siNDUFA4L2 for 24h and then treated with 100 μM of ascorbic acid 2-phosphate (AA2P) for 24h. After several washes to remove AA2P, the cells were incubated for 18 h in normoxic or hypoxic conditions. Lastly, the cells were trypsinized and the viable cells were counted using trypan blue.
To study mitochondrial superoxide generation, Caki-2 cells were seeded at a density of 2x10⁵ on glass coverslips and left to adhere overnight at 37°C in 5% CO2. The next day the cells were washed and incubated with siNDUFA4L2 and AA2P, as described above. Caki-2 cells were stained with 5 μM MitoSOX red for 10 min at 37°C and washed three times before imaging. Caki-2 cells were fixed using ice-cold 4% paraformaldehyde for 10 min at room temperature. Then, nuclei were revealed by counterstaining with TO-PRO-3 (Molecular Probes). Images were taken with a Leica TCS SP2 (Leica, Wetzlar, Germany) confocal laser-scanning microscope using an X63 objective lens. Each experimental condition was performed in triplicate.