Down-regulation of DAPK in THP-1 decreases caspase-1 activation and p17 IL-1β generation, but not TNF-α production. (A) DAPK knockdown did not affect the expression of NLRP3, ASC, and procaspase-1. THP-1 cells were infected with lentivirus (pLL3.7) containing DAPK-specific shRNA (shDAPK) or control shRNA (shCtrl), the infected cells were sorted, and the contents of DAPK, NLRP3, ASC, and procaspase-1 determined by immunoblots. (B-C) Down-regulation of DAPK decreased secretion of IL-1β but not TNF-α. Control and DAPK-knockdown THP-1 cells, pretreated with PMA (500 ng/mL) for 3 hours and cultured overnight, were stimulated with or Pam3CSK (Pam3, 0.5 μg/mL) or crude LPS (15 μg/mL) for 5 hours, MSU (150 μg/mL) for 6 hours, after which cells and supernatants were collected. The levels of IL-1β (B) and TNF-α (C) in supernatants were determined by ELISA. (D-F) Reduced generation of p17 IL-1β and p10 caspase-1 in DAPK-knockdown THP-1 cells. Cell supernatants (SUP) from panel B were precipitated by cold acetone and analyzed, together with total cell lysates (TCL) from panel B, by SDS-PAGE. The contents of pro-IL-1β, active IL-1β, procaspase-1, active caspase-1, and GAPDH in THP-1 stimulated with MSU (D), Pam3CSK (E), or crude LPS (F) were determined by blotting with specific antibodies. Error bar represents SD of a specific experiment of triplicate samples. All experiments have been repeated 3 times with similar results. **P < .01, ***P < .001 for paired t test.