Fig 1.
Inhibition of DNA-PKcs in T cells and PBMCs blocks IL-2 production.
A) Jurkat cells were treated with the DNA-PKcs inhibitor NU7441 at varying concentrations for 48 hours and no significant reduction in viability was detected. B) Jurkat cells were stimulated with PMA (50 ng/mL)+PHA (1 μg/mL), treated with NU7441, and analyzed for IL-2 production 24 hours later. NU7441 treatment significantly blocked IL-2 secretion. C) IL-2 production stimulated by activation of Jurkat cells with anti-CD28/CD3 dynabeads at a 1:1 ratio for 24 hours was inhibited by NU7441 treatment. D) Treatment of Jurkat cells with shRNA reduced DNA-PKcs expression at 2.5 and 5 μg as seen by western blot analysis. Loss of DNA-PKcs expression significantly reduced IL-2 production. E) NU7441 significantly reduce IL-2 production following activation with PHA+PMA in PBMCs. ** p< 0.002 *** p<0.001 error bars = s.d.
Fig 2.
Inhibition of DNA-PKcs blocks translocation of NFAT to the nucleus.
A) Western blot analysis of Jurkat cell lysates showed activation of T cells with PMA+PHA induced phosphorylation of DNA-PKcs at site s2056 (pDNA-PK) and dephosphorylated NFAT at s237 (pNFAT). Treatment with NU7441 inhibited the dephosphorylation of NFAT at site s237 which is critical for its translocation to the nucleus. GAPDH was used as a loading control. B) Immunocytochemistry analysis of Jurkat cells treated with NU7441. The inhibitor (2.5 μM) blocked translocation of NFAT to the nucleus following activation with PMA+PHA. Nuclei were stained with Dapi. 40X images are shown.
Fig 3.
DNA-PKcs inhibition blocks calcineurin activity in T cells.
A) Jurkat cells were activated with PMA+PHA, treated with the DNA-PKcs inhibitor NU7441 (2.5μM) and monitored for calcineurin phosphatase activity. Inhibition caused a significant reduction in calcineurin activity. B) Level of Ca2+ in Jurkat cell lysates following activation with PMA+PHA was monitored. Ca2+ levels were not affected by the addition of the NU7441 inhibitor. C) Western blot and Elisa analysis of active phosphorylated mTOR in activated Jurkat cells indicated that inhibition of DNA-PKcs does not alter mTOR activation. ***p<0.001 error bars = s.d.
Fig 4.
Inhibition of DNA-PKcs reduces phosphorylation of CHK2 and stabilizes the calcineurin inhibitor, Cabin1.
A) Western blot analysis of Jurkat lysates following activation with PMA+PHA and NU7441 treatment. Activation increased phosphorylation of DNA-PKcs and CHK2. DNA-PKcs inhibition reduced CHK2 phosphorylation and elevated Cabin1 expression. GAPDH was used as a loading control. B) Schematic depicting the signaling pathway in T cells used by DNA-PKcs to regulate IL-2 production. DNA-PKcs phosphorylates CHK2 which in turns phosphorylates Cabin1 targeting it for destruction. This alleviates calcineurin inhibition causing an increase in translocation of NFAT and IL-2 production. CaN, calcineurin.