After washing and 1 hr incubation at RT with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:2,000; Cell Signaling Technologies), immunoreactive Deforolimus
proteins were detected with an enhanced chemiluminescence system (Millipore Corporation, Billerica, MA). ��-Tubulin was probed as loading control using mouse monoclonal anti-��-tubulin primary antibody (1:5,000; Millipore Corporation, Temecula, CA) and horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:5,000; Millipore Corporation). Exponentially growing cells were dissociated and single cell suspensions were prepared in regular DMEM to be seeded in 6-well plates at a density of 100 cells per well. After 6 hr of incubation to allow cell adherence, supernatants were removed, cells were carefully washed with PBS and specific treatment media were added (2 ml/well). Canavanine at 0.1 mM in complete DMEM did not affect cell proliferation and was not removed throughout culturing (i.e., 17 days). Arginine-free culture medium both with and without canavanine interfered with cell proliferation and thus selleck chemicals
treatment in colony formation assays was limited to 1 or 3 days after which the medium was supplemented with 0.4 mM arginine. Here, cells were allowed to grow for additional 17 days. Accordingly, irradiation with an YxlonY.TU 320 device (1.3 Gy/min, 200 kV X-Rays, 0.5 mm Cu filter; Yxlon International, Hamburg, Germany) was performed at RT 6 hr after seeding for cells cultured in complete medium (CM) with/without canavanine, and after 1 or 3 days if cells were exposed to arginine-free medium with/without canavanine. Colonies were stained with 0.5% crystal violet followed by manual microscopic counting of colonies with >50 cells and calculation of plating efficiencies (PEs) and survival fractions (SFs). The experiment was performed in triplicate with a minimum of three individual wells per condition ADAMTS5
in each experiment. After initiation, spheroids were collected, washed with PBS and transferred to new 96-well plates as described in Growth restoration experiments. The experimental design included single dose irradiation at RT with 0, 5, 7, 10, 12, 15, 17 and 20 Gy (YxlonY.TU 320) at a dose rate of 1.3 Gy/min. Irradiation was performed at day 1 or 5 of treatment. In all cases, incubation in the respective media lasted 5 days and then arginine-free media were supplemented with arginine to the final concentration of 0.4 mM. Further medium renewal was performed with regular DMEM. Spheroids were monitored up to 60 days in culture. A minimum of 32 spheroids per treatment condition were analyzed. Data are presented as Mean �� SD. All p values were calculated by two-sided Student's t-test. The difference was considered to be statistically significant at the level of p < 0.05.