Figure 5: 25-HC activates the NLRP3 inflammasome signalling in a potassium efflux/mitochondrial ROS/LXR-dependent manner. | Nature Communications

Figure 5: 25-HC activates the NLRP3 inflammasome signalling in a potassium efflux/mitochondrial ROS/LXR-dependent manner.

From: 25-hydroxycholesterol contributes to cerebral inflammation of X-linked adrenoleukodystrophy through activation of the NLRP3 inflammasome

Figure 5

(a) Immunoblots of mouse BMDMs treated with 25-HC (50 μM, 10 h) and LPS (final 4 h) in the presence of glibenclamide (50 μM) or KCl (20 mM) for the indicated times, or treated with LPS, followed by ATP. (b) Immunoblots of BMDMs treated with 25-HC (50 μM, 10 h) and LPS (final 4 h) in the presence of glibenclamide (50 μM, 10 h). (c) PL assay of NLRP3 and ASC in microglia similarly treated as in b. Relative PL signals (per DAPI) are displayed. (n=5) *P<0.05 (d) Flow cytometric analysis of BMDMs treated with 25-HC (50 μM, 10 h) and LPS (final 3 h) after staining with MitoSOX (upper panel) or co-stained with MitoTracker Green and MitoTracker Deep Red (lower panel). (e,f) Immunoblots of BMDMs primed with LPS (3 h), followed by treatments with 25-HC (80 μM) in the presence of NAC (20 mM) or Mito-TEMPO (MT, 200 μM) for 6 h. (g,h) Immunoblots of BMDMs primed with LPS (3 h), followed by 25-HC (80 μM) in the presence of 22-HC (50 or 100 μM) for 6 h (g), or primed with LPS in the presence of 22-HC (100 μM) or glibenclamide (50 μM) for 3 h, followed by ATP (2 mM, 45 min) (h). (i) Immunoblots of BMDMs primed with LPS (3 h), followed by 25-HC (80 μM) in the presence of 22-HC (100 μM) or glibenclamide (50 μM) for 6 h. (a,b,e–i) Culture supernatants (Sup), cellular lysates (Lys) or DSS-crosslinked pellets (Pel + DSS) as indicated were analysed by immunoblot.

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