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. 2022 Aug 24;10(8):e004832. doi: 10.1136/jitc-2022-004832

Figure 3.

Figure 3

The miR-21-containing exosomes represses BRCC and PTEN to reduce NLRP3 inflammasome activity. (A) Immunoprecipitation (IP)-western blots to show the interaction of NLRP3 and ASC in HEK293T cells transfected with Flag-NLRP3, GFP-ASC and miR-21/control vector. IgG is a control for IP. (B) Immunoblots for examining the expression of PTEN in macrophages derived from THP-WT or THP1MIR21–/–. (C) IP-western blots to show the tyrosine phosphorylation of THP-WT/THP1MIR21–/–-derived macrophages primed by LPS (1 µg/mL) and activated by nigericin (5 µM). IgG is a control for IP. (D) Left upper, western blot of BRCC3 in THP1 cells receiving short hairpin RNA against BRCC3. Left lower, western blots of BRCC3 in THP-WT/THP1MIR21–-derived macrophages. α-tubulin was a loading control. Right, quantitative real-time PCR for examining the relative expression of BRCC3 in THP-WT/THP1MIR21–/–-derived macrophages. n=3 (each contains two technical replicates). Data shows mean±SD. **p<0.01 by Student’s t-test. (E) BRCC3 3’-UTR reporter assay. The wild-type or miR-21 binding site mutated 3’-UTR reporter constructs of BRCC3 (pMIR-BRCC3-wt and pMIR-BRCC3-mut), pcDNA3-miR21/control vector and β-galactosidase were co-transfected to HEK293T cells. Data represent means±SD. **p<0.01 by Student’s t-test. n=3 independent experiments (each contains two technical replicates). (F) IP-western blot to show the K63-ubiquitylated NLRP3 in HEK293T cells transfected with miR-21 or a control vector. NLRP3 (left panel) or HA-tagged K63 ubiquitin (right panel) was immunoprecipitated for immunoblotting. IgG is a control for IP. (G) IP-western blot to show the K63-ubiquitylated NLRP3 in THP1-derived activated macrophages primed by LPS (1 µg/mL) and transfected with miR-21 or a control vector. Nigericin (5 µM) was used to activate inflammasome. IgG is a control for IP. (H) IP-western blot to show the K63-ubiquitylated NLRP3 in THP1-WT/THP1MIR21–/–derived activated macrophages. IgG is a control for IP. miR, micro RNA; UTR, untranslated region.