SATB1-dependent mitochondrial ROS production controls TCR signaling in CD4 T cells

(A) CD4 T cells were suspended in ATP assay buffer and incubated for 1 min at RT. Light intensity was indicative of intracellular ATP content (Lu-ATP). (B) Oxidative phosphorylation complex I enzyme activity was measured. Lysates were incubated on the plate for 3 h to capture complex I. Enzyme activity was determined by NADH oxidation to NAD+ and simultaneous dye reduction. Dye absorbance was measured with a plate reader at 450 nm. (C) Levels of mtROS in CD4 T cells from WT, and SATB1cKO mice in the control and after TCR activation were measured by DCFDA staining and flow cytometry. (D) Quantification of mean fluorescence of DCFDA. (E) Representative MitoTracker staining histograms. WT and SATB1cKO T cells in the control and after TCR activation were stained with MitoTracker and analyzed by a flow cytometer. (F) Quantification of the mean fluorescence of MitoTracker.*P < 0.01 versus WT. N = 5. Data are shown as the means ± SD.

WT and SATB1cKO naïve CD4 T cells were stimulated with anti-CD3 and anti-CD28 antibodies for the indicated times and lysed in sample buffer. Oxidized proteins in the cell lysates were labeled with iodoaceto-PEG biotin and immunoprecipitated. (A) Blots were probed for anti–SHP-1 antibody. (B) Band intensities were quantified with ImageJ v. 1.49. (C) Naïve CD4 T Cells were incubated for 0, 10, 20, 30, 60, and 120 min with anti-CD3 and anti-CD28 antibodies and lysed in phosphatase assay buffer. Phosphatase activity was assayed by incubation with PY containing peptide and liberated phosphate was measured by malachite green absorption at 450 nm. (D, E) Lysates of SATB1-deficient naïve CD4 T cells were incubated in the absence or presence of H₂O₂ for 15 min. Oxidized SHP-1 was detected by immunoblotting with antibody against SHP-1 (D). (E) Band intensities were quantified with ImageJ v. 1.49 (E). (B, F) Phosphatase activity in the presence or absence of H₂O₂ was measured in (B). Data are shown as the means of three experiments performed in duplicate. *P < 0.01 versus WT. **P < 0.05 versus WT. N = 5. Data are shown as the means ± SD.

(A) Representative MitoTracker staining histograms. WT and SATB1cKO T cells were stained with MitoTracker and analyzed by FACS. (B) Quantification of the mean fluorescence of MitoTracker is shown. (C) Mitochondrial DNA copy numbers in CD4 T cells from WT, SATB1 cKO, TFAMcKO, and SATB1cKO-TFAMTg mice were measured by RT-qPCR. The mitochondrial DNA level was normalized to the nuclear DNA level in each sample. The normalized index is indicated as an arbitrary unit (WT = 1). (D) TFAM and β-actin levels in CD4 T-cell lysates were analyzed by immunoblotting using the respective antibodies. (E) CD4 T cells were prepared from WT and TFAMcKO mice, stimulated for the indicated times with anti-CD3 and anti-CD28 antibodies, lysed, analyzed by SDS–PAGE, and immunoblotted with antibodies against phospho-ZAP70, and ZAP70. Similar results were obtained for three independent experiments. *P < 0.01 versus WT. **P < 0.01 versus SATB1cKO T cells. N = 5. Data are shown as the means ± SD.