Inhibition of xCT with sulfasalazine decreases proliferation and increases apoptosis of SPEM cells. (A) ImSPEM cells were seeded at the same density ± sulfasalazine for 3 days. Representative transmitted light images monitoring cell density over 3 days. Scale bar: 100 μm. (B) ImSPEM cell number ± sulfasalazine at 24, 48, and 72 hours (P = .001∗, .006∗, and .0002∗, respectively). (C) ImSPEM cells were treated with sulfasalazine for 48 hours and immunostained for proliferation marker Ki67 (red) with nuclear counterstain 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar: 100 μm. (D) Percentage of cells per 20× field that are Ki67 positive (P = .01∗). (E) ImSPEM cells were treated with sulfasalazine for 72 hours and immunostained for apoptosis marker cleaved caspase-3 (CC3) (green) with nuclear counterstain DAPI (blue). Scale bar: 100 μm. (F) Percentage of cells per 20× field that are CC3 positive (P = .003∗∗). Statistical significance was determined by unpaired Student t test (n = 3 per condition).