Fig. 3.

DLD regulates ferroptosis induced by cystine deprivation or import inhibition. (A–C) Cell death and lipid ROS assays in HN9 cells stably transduced with shRNA targeting DLD or a vector control (vtr). The cells were subjected to CC deprivation or 1 mM sulfasalazine (SAS) treatment for 48 h (PI-positive cells) or 8 h (BODIPY fluorescence). Scale bar, 50 μm **P < 0.001 relative to vtr or control without vector or shDLD (ctr). (D–F) Cell death and lipid ROS in HN9 cells stably transduced with a resistant DLD cDNA (DLDres), shDLD, or a vector control. PI and BODIPY were stained and counted using flow cytometry after the cells were subjected to CC deprivation or 1 mM SAS treatment for 48 h and 8 h, respectively. Western blots were performed in these cells stably transduced with DLDres, shDLD, or vector. **P < 0.001 relative to a vector control. (G–J) Effects of DLD gene silencing on SAS-induced inhibition of tumor growth in vivo. Tumor volumes were regularly measured after SAS or vehicle treatments in nude mice implanted with shDLD or vtr-transduced HN9 cells. Lipid ROS level and mitochondrial iron accumulation in tumors were measured using BODIPY and rhodamine B-[(1,10-phenanthroline-5-yl)-aminocarbonyl]benzyl ester (RPA). *P < 0.01 relative to control. NS indicates statistically not significant.