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. 2024 Dec 17;7:0551. doi: 10.34133/research.0551

Fig. 5.

Fig. 5.

HSP90α inhibition directly disrupts cage-NLRP3. (A) BMDMs were treated with 20 μM MG132 and 30 μM CQ for 2 h, and then 100 ng/ml LPS for 3 h followed by 10 μM polydatin or 10 μM GA treatment for 1 h. HEK293T cells transfected with flag-tagged NLRP3 or BMDMs were treated with 20 μM MG132 and 30 μM CQ for 2 h, followed by 10 μM polydatin or 10 μM GA treatment for 1 h. The cells were then treated with 40 μg/ml digitonin on ice to permeabilize the plasma membrane. (B) The NLRP3 monomers and double-ring cage-NLRP3 oligomers were separated and examined by immunoblot. (C) BMDMs were stimulated with 100 ng/ml LPS for 3 h (above). HEK293T cells were transfected with flag-NLRP3 (below). The cells were treated with 40 μg/ml digitonin on ice to permeabilize the plasma membrane. (D) The pellets were treated with 10 μM polydatin or 10 μM GA for 1 h, and the NLRP3 expression was analyzed by immunoblot. (E) BMDMs stimulated with 100 ng/ml LPS were treated with 10 μM polydatin or 10 μM GA treatment for 1 h. NLRP3 expression was examined using IF. (F) HEK293T cells transfected with GFP-NLRP3 were treated with 10 μM polydatin or 10 μM GA. GFP expression was observed by confocal microscopy. (G) The stable HSP90α KO HEK293T cell line was conducted by CRISPR-Cas9. HSP90α KO HEK293T cells were transfected with GFP-NLRP3 and mCherry-HSP90α and then observed by confocal microscopy. (H and I) HEK293T cells transfected with flag-NLRP3 were cotransfected with GFP-HSP90α or infected with HSP90α Cas9 lentivirus. The expression of HSP90α and NLRP3 was determined by immunoblot. (J) An endogenous IP with HSP90α antibody was performed in LPS-primed BMDMs treated with or without CQ and MG132 in the presence of polydatin and GA. Western blot analysis of HSP90α and NLRP3. (K) HEK293T cells were transfected with flag-NLRP3 and GFP-HSP90α and treated with or without CQ and MG132. Interaction between HSP90α and NLRP3 in the presence of polydatin and GA was examined by IP. Data are representative of 3 independent experiments. Scale bar, 10 μm.