HPK1 enhances apoptosis of EL-4 cells by ROS.

(A) HPK1 is up-regulated and cleaved upon induction of H₂O₂-mediated apoptosis in EL-4 cells. EL-4 cells were stimulated with 1 mM H₂O₂ for 0 to 120 minutes. Whole cellular proteins (100 μg) were resolved by SDS-PAGE, immunoblotted, and detected with Abs specific for HPK1 (which indicates full-length HPK1, above, or the N-terminal cleavage product) or β-actin. Representative blots from 3 independent experiments are shown. (B) HPK1 promotes H₂O₂-mediated apoptosis of EL-4 cells. EL-4 cell lines transduced with pEGZ-HA, pHA-HPK1 wt, or pHA-HPK1 AS viruses were left either untreated or treated with 1 mM H₂O₂ for 2 to 8 hours. Cell-cycle profiles were determined using propidium iodide staining by FACS analysis. SubG1 phase cells were identified as apoptotic cells. The graph shows the fold up-regulation of apoptosis of cells expressing HPK1 (or HPK1 AS) compared to that of control cells transduced with pEGZ-HA. The results presented are the mean values of 5 independent experiments. In the inset (above, left), an immunoblot is shown, which demonstrates the suppressive effect of human HPK1-AS RNA on the expression of endogenous murine HPK1. Whole protein extracts (80 μg) were fractionated from EL-4 cells stably infected with control pEGZ-HA (lane 1), pHA-HPK1 wt (lane 2), or pHA-HPK1 AS viruses (lane 3). Below, the expression of β-actin is shown as loading control. (C) HPK1-AS RNA strongly reduces HPK1 expression. Human 293T cells were cotransfected with retroviral DNAs. After 48 hours, cells were lysed, 50 μg proteins was resolved by SDS-PAGE and immunoblotted with Abs specific for HPK1 or β-actin. Representative blots from 3 independent experiments are shown.