Figure 3.

Bortezomib conditioning reduced bortezomib sensitivity and increased xCT and GSH levels in INA-6 cells. (a) Bortezomib-conditioned (Bz-cond.) or non-conditioned (Non-cond.) INA-6 cells were treated with the indicated doses of bortezomib (Bz.) for 24 h. Cell death was measured using the ScanR microscope, as described in Materials and methods. The results represent the mean and s.d. of three independent experiments. Bortezomib conditioning significantly increased the IC50 (nm) of bortezomib. (b) RNA was collected from bortezomib-conditioned and non-conditioned INA-6 cells and analyzed for mRNA levels of SLC7A11 by qPCR. Data are mean and s.d. from three independent experiments. The asterisk indicates a statistically significant difference as compared with the control (P<0.05, Student's t-test). (c) Bortezomib-conditioned and non-conditioned INA-6 cells were lysed and baseline xCT levels were analyzed by immunoblotting. GAPDH was used as a loading control. Data are representative for at least three independent experiments. (d) Bortezomib-conditioned and non-conditioned INA-6 cells were treated with the indicated doses of hydrogen peroxide (H₂O₂) for 16 h. Cell death was measured using the ScanR microscope. Bortezomib conditioning significantly increased the IC50 (μm) of hydrogen peroxide. Data display mean and s.d. of duplicates from one representative of three independent experiments. (a, d) IC50 was calculated using non-linear regression. Extra sum-of-squares F-test was used to test for significant shifts in IC50 (P<0.05).