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. 2021 Jan 28;69(6):1413–1428. doi: 10.1002/glia.23970

FIGURE 4.

FIGURE 4

(a) Primary microglia from WT mice show an IL‐1β secretion response to 16 hr exposure to 144.6 μg/ml α‐syn, while microglia from NLRP3−/− mice do not. (b) Il‐1β secretion from WT primary mouse microglia induced by exposure to 144.6 μg/ml α‐syn for 16 hr is abrogated by inhibition of either NLRP3 with MCC950 or caspase‐1 activity with Z‐YVAD‐FMK (YVAD). (c) Primary microglia from WT mice demonstrate an increase in NLRP3 protein levels in cell lysates as detected on western blot in response to the same treatment as in Figure 4a, while microglia from NLRP3−/− mice do not. Spliced to juxtapose NLRP3 bands directly. (d) MCC950 Inhibition of LPS or α‐syn‐induced IL‐1β secretion by the THP‐1 model. Four experiments, n = 2 or 3. (e) LPS‐ or α‐syn‐induced IL‐1β secretion from THP‐1 cells is partially dependent on caspase‐1 activity. Four experiments, n = 2 or 3. (f) MCC950 Inhibition of LPS‐ or α‐syn‐induced IL‐1β secretion by primary human microglia. Two experiments, n = 3. (g) LPS‐ or α‐syn‐induced IL‐1β secretion from primary human microglia is independent of caspase‐1 activity. 2 (LPS) or 3 (α‐syn) experiments, n = 3. Colors indicate individual experiments. (h,l) NLRP3 western blots of combined, concentrated supernatants of several primary human microglia experiments after canonical (h) or LPS‐ or α‐syn‐mediated (l) NLRP3 inflammasome activation. (i,m) Quantification of NLRP3 bands shown in h (I, canonical activation) and l (m, LPS‐ or α‐syn‐mediated activation). (j,n): Caspase‐1 western blots of combined, concentrated supernatants of several primary human microglia experiments after canonical (j) or LPS‐ or α‐syn‐mediated (n) NLRP3 inflammasome activation. (k,o) Quantification of caspase‐1 bands shown in j (k, canonical activation) and n (o, LPS‐ or α‐syn‐mediated activation). (p) Caspase‐1 western blot of lysates of THP‐1 cells after canonical, LPS‐, or α‐syn‐mediated NLRP3 inflammasome activation versus β‐actin loading control. ns, not significant, **** = p <.0001 [Color figure can be viewed at wileyonlinelibrary.com]