Figure 3.

ROP7 in THP-1 cells activated NLRP3 inflammasome and induced a hyperactive status in cells. THP-1_Mock and THP-1_ROP7 were induced by 1-μg/mL doxycycline for 48 h and differentiated under 100-nM PMA for another 48 h. THP-1 infected with lentivirus as a mock control, THP-1_Mock. (A) NLRP3 and ASC were co-precipitated with Flag-HA-ROP7 in THP-1 cells. After expression and differentiation, the cells were harvested for Co-IP using an anti-HA antibody to immunoprecipitated Flag-HA-ROP7 and further analyzed by Western blot using an anti-NLRP3, anti-ASC, and anti-Flag antibody. (B) After differentiation, cell medium was renewed and collected periodically to detect IL-1β and TNF-α secretion by ELISA. Data are expressed as mean ± SEM values. * p < 0.05, *** p < 0.001, as compared with THP-1_Mock. (C) pro-IL-1β and p17 of THP-1_Mock and THP-1_ROP7 in cell lysate and supernatant (SN) were detected by immunoblotting using anti-IL-1β. In total, 100-ng/mL LPS was used in THP-1_Mock for 4 h as a positive control. (D) At 24 h post-induction, the cells were treated with 100-ng/mL LPS for 4 h alone or 10-μM nigericin for 1 h subsequently. Supernatants were collected for ELISA. Data are expressed as mean ± SEM values. ** p < 0.01, *** p < 0.001, as compared with THP-1_Mock. (E) After PMA differentiation, 10-μM of Z-YVAD-FMK, Z-VAD-FMK, or MCC950 was added to the cells for 24 h. The IL-1β and TNF-α in cell-free supernatant were detected by ELISA. Data are expressed as mean ± SEM values. *** p < 0.001, as compared with the DMSO group. (F) Supernatants that were untreated (24 h after PMA differentiation and medium replacement), LPS-treated (100 ng/mL for 4 h), and nigericin-treated (10 μM for 1 h) were collected for the detection of LDH release. The results were normalized by the maximum enzyme activity in each group. Data are expressed as mean ± SEM values. ns: non-significant, ** p < 0.01.