Figure 5. IGF-I regulated intracellular ROS level via xC⁻ transporter and through PI3K dependent pathway in ER positive breast cancer cells.

(A) MCF-7 and T47D cells were pretreated with or without SASP (0.1 mM) for 48 h, grown in SFM for 24h, treated with IGF-I (5 nM) for another 24h, and then irradiated (10 Gy, left panel) or treated with mitomycin C treatment (1 μg/ml for ROS assay; 0.1 μg/ml for immunoblot) for 3 days (right panel). Intracellular ROS levels were measured as described. (B) Cellular phospho-p38^MAPK level was determined by immunoblot. (C) MCF-7 cells were grown in SFM, pretreated huEM164 (20 μg/ml), LY294002 (10 μM), or NVP AEW-541 (0.5 μM) (Left panel). MCF-7 cells were pretreated with indicated dosage of SASP, BSO, or NAC. Acute ROS were induced by 5 gy irradiation. MCF-7 cells incubated with ROS insensitive probe carboxy-DCFDA (0.1 mM) was presented as assay control (Right panel). Cellular ROS levels were determined as described. (D) MCF-7 cells were treated with indicated treatments in soft agar. After 24h of synchronization in SFM condition, cells were either irradiated or treated with mitomycin C. Colony formation was assessed after 14 days. Survival rate curve was determined by normalizing colony number of each treatment to its own no treatment control. Statistically significant differences are noted (* or # p<0.5, ** or ## p<0.01, *** or ### p<0.001). IC₅₀ values were shown in the table. Data are mean ± SEM; all results are representative of three independent replicates.