Fig. 1.

Signaling pathways that regulate the early-phase generation of ROS in TCR-stimulated Jurkat cells. (A) Kinetics of TCR-induced generation of ROS in Jurkat cells detected within the indicated intervals by measurement of the oxidation of DCFDA (open circles) or DHE (solid squares). (B) TCR-induced oxidation of DCFDA measured in 10-min endpoint assays after stimulation with antibody against CD3 in control Jurkat cells (E6-1) or in Jurkat cell clones in which Lck (J.CaM1) or ZAP-70 (P116) is absent. Cells were stimulated by antibody against CD3 (OKT3) cross-linked by secondary antibodies (open bars) or with PMA (10 ng/ml) and ionomycin (1 μg/ml; solid bars). (C) TCR-induced oxidation of DCFDA in wild-type (WT) Jurkat cells (E6-1), in clones that are deficient in IP₃R1 (IP₃R1-deficient) or PLC-γ1 (J.gamma1), or in a PLC-γ1–deficient clone that was reconstituted with PLC-γ1 (J.gamma1.WT). *P < 0.05, when compared to Jurkat E6-1 cells; ^#P < 0.05, when compared to J.gamma1 cells. (D) TCR-induced oxidation of DCFDA in Jurkat cells that were transiently transfected with empty vector or with plasmids encoding either WT PLC-γ1 or the Y783F mutant PLC-γ1 in the presence of a plasmid encoding RFP. The data represent the percentage of TCR-stimulated oxidation of DCFDA in RFP⁺ cells. (E) TCR-induced oxidation of DCFDA in WT Jurkat cells or in SLP-76–deficient clones (J14) that were stably transfected with plasmids encoding full-length SLP-76 (J14-WT), a tyrosine mutant of SLP-76 (J14-Y3F), an SLP-76 mutant in which the proline-rich domain was deleted (J14-ΔGads), or a variant SLP-76 in which the SH2 domain is mutated (J14-mSH2). (F) TCR-induced oxidation of DCFDA in WT Jurkat cells in the presence or absence of the Ca²⁺ chelators EGTA and BAPTA or in cells stimulated in buffer lacking Ca²⁺ (−Ca²⁺). In all of the panels, the data represent means ± SEM of at least four separate experiments. *P < 0.05.