Figure 5. VHH ASC prevents activation of... | Rockefeller University Press

Figure 5.

$Figure 5. VHHASC prevents activation of NLRP3 and AIM2 inflammasomes in THP-1 cells. (A) THP-1 cell lines inducibly expressing VHHASC-HA or VHH NP1-HA were cultivated for 24 h in the absence or presence of doxycycline, fixed, stained for HA, and analyzed by flow cytometry. (B–E) Wild-type THP-1 cells and the cell lines described in A were differentiated and VHH expression induced for 24 h. (B) Cells were treated with 200 ng/ml LPS for 3 h, harvested, and cell lysates analyzed by immunoblot with anti-ASC, anti-HA, and anti-GAPDH. (C) Cells were treated with 200 ng/ml LPS for 4 h or left untreated. Supernatants were analyzed by TNF ELISA. (D and E) Cells were treated with LPS for 3 h and nigericin for 45 min, or left untreated. Supernatants were analyzed by IL-1β ELISA (D) and by immunoblots with anti–IL-1β and anti-caspase-1 p10 (E). Immunoblots representative of three experiments are shown. The asterisk indicates a statistically significant difference (Student‘s t test; P < 0.001). (F and G) Cells treated as in D were fixed and stained for DNA and ASC; images were recorded by wide field fluorescence microscopy (F) and the fraction of cells containing ASC foci was quantified (G). Data from three independent experiments with at least 500 cells per condition ± SEM is shown. Bars, 20 µm. (H) THP-1 cell lines cultivated as described in B were treated with LPS for 3 h and transfected with poly (dA:dT) or transfection agent only. Supernatants were harvested after 4 h and IL-1β levels were quantified by ELISA. The asterisk indicates a statistically significant difference (Student‘s t test; P = 0.002). Data from three independent experiments was quantified for all ELISA results and mean values ± SEM are shown.$

VHH_ASC prevents activation of NLRP3 and AIM2 inflammasomes in THP-1 cells. (A) THP-1 cell lines inducibly expressing VHH_ASC-HA or VHH NP1-HA were cultivated for 24 h in the absence or presence of doxycycline, fixed, stained for HA, and analyzed by flow cytometry. (B–E) Wild-type THP-1 cells and the cell lines described in A were differentiated and VHH expression induced for 24 h. (B) Cells were treated with 200 ng/ml LPS for 3 h, harvested, and cell lysates analyzed by immunoblot with anti-ASC, anti-HA, and anti-GAPDH. (C) Cells were treated with 200 ng/ml LPS for 4 h or left untreated. Supernatants were analyzed by TNF ELISA. (D and E) Cells were treated with LPS for 3 h and nigericin for 45 min, or left untreated. Supernatants were analyzed by IL-1β ELISA (D) and by immunoblots with anti–IL-1β and anti-caspase-1 p10 (E). Immunoblots representative of three experiments are shown. The asterisk indicates a statistically significant difference (Student‘s t test; P < 0.001). (F and G) Cells treated as in D were fixed and stained for DNA and ASC; images were recorded by wide field fluorescence microscopy (F) and the fraction of cells containing ASC foci was quantified (G). Data from three independent experiments with at least 500 cells per condition ± SEM is shown. Bars, 20 µm. (H) THP-1 cell lines cultivated as described in B were treated with LPS for 3 h and transfected with poly (dA:dT) or transfection agent only. Supernatants were harvested after 4 h and IL-1β levels were quantified by ELISA. The asterisk indicates a statistically significant difference (Student‘s t test; P = 0.002). Data from three independent experiments was quantified for all ELISA results and mean values ± SEM are shown.