Fig. 3.
H2S improves the stability of xCT through persulfidation of OTUB1 at cysteine 91. (A) Cycloheximide chase assays showing decreased stability of xCT after inhibiting xCT, which was ameliorated by 500μMGYY4137 (* P < 0.05 vs CTH siRNA). (B) Modified biotin switch assays showing decreased persulfidation of OTUB1 after inhibiting xCT, which was ameliorated by GYY4137. (C) Immunoblots following IP showing decreased expression of xCT and decreased conjunction between xCT and OTUB1 after inhibiting CTH in HCT-116 cells, which was ameliorated by GYY4137. (D) IF of xCT indicating decreased xCT expression after inhibiting xCT, which was ameliorated by GYY4137. (E) Modified biotin switch assays showing no effect of these expressing plasmids on the persulfidation mediated by GYY4137 in HCT-116 cells, dithiothreitol (DTT) treated samples served as negative controls. (F) Immunoblots showing that OTUB1 C91A expressing plasmids abrogated the increased xCT levels induced by GYY4137 in HTC-116 cells transfected with exogenous OTUB1 expressing plasmids and OTUB1 siRNAs. (G) Immunoblots after IP showing no effect of OTUB1 C23A plasmids on the increased persulfidation and increased conjunction between OTUB1 and xCT induced by GYY4137. (H) OTUB1 C91A plasmids abrogated the increased persulfidation and increased conjunction between OTUB1 and xCT induced by GYY4137.