O₂⁻ promotes chemoresistance through the induction of S70 Bcl-2 phosphorylation. (A) Bcl-2 phosphorylation status was examined in Jurkat cells treated with increasing doses of DDC for 4 hours. A total of 400 μM was selected as the working dose for subsequent experiments. (B) Western blot of Jurkat cells after genetic silencing of SOD1 using 4 independent siRNA sequences (denoted as siSOD1-A, B, C, and D). (C) S70pBcl-2 level was assessed in Jurkat cells transfected with vector (EV) or SOD1 plasmids. (D) Immunoblot of Jurkat cells treated with bovine SOD1 (bSOD1). bSOD1 appeared as faster migrating band under human SOD1. (E) Jurkat cells were pretreated for 1 hour with tiron, followed by 4 hours with DDC, and then lysates were immunoblotted for S70pBcl-2. (F) MTT cell viability assay of Jurkat cells pretreated with DDC (1 hour) followed by treatment with the indicated doses of chemotherapeutic agents (5FU, cisplatin, etoposide [Eto], and doxorubicin [Doxo]) for 18 hours. (G) MTT assay of Eto- or Doxo-treated Jurkat cells after knockdown of SOD1 using siSOD1A, B, or C. (H) MTT assay of 5FU- or cisplatin-treated Jurkat cells after pcDNA-SOD1 transfection or after the inhibition of reduced NADP oxidase-mediated O₂⁻ production via DPI (2.5 μM) pretreatment. (I) MTT assay of Jurkat cells expressing wild-type Bcl-2, S70A Bcl-2, or S70E Bcl-2 mutant after 18 hours of Eto treatment.