Fig 2: XPJYF treatment targeted astrocyte activation and glucose metabolism. (A) Uptake of 2DGP in astrocyte. (B) Glucose‐1‐phosphate level in astrocyte. (C) ATP content. (D) Western blotting. (E) The relative expression of COX‐2 in astrocyte. (F) IL‐6 expression in astrocyte. (G) TNF‐α expression in astrocyte. (H) iNOS expression in astrocyte. (I) IL‐1 expression in astrocyte. (J) BDNF expression in astrocyte. (K) Astrocyte activation by GFAP immunofluorescence.

Fig 3: A20 is a functional miR-21 target for regulation of NF-?B and NLRP3 inflammasomes.a–e A20 (a), Nlrp3 (b), Asc (c), Casp1 (d), and IL-1b (e) mRNA levels in LPS-treated WT and miR-21 KO BMDMs. f A20, IL-1b and NLRP3 inflammasome protein levels in LPS-treated WT and miR-21 KO BMDMs after 4 h. g Sequence alignment of the A20 3'-UTR with miR-21 across multiple species. h Activity of luciferase reporters containing WT or mutant A20 3'-UTRs that were used with miR-21 mimics or respective controls to cotransfect HEK-293T cells. i BMDMs were treated with 500 ng/ml LPS for different lengths of time. Cell extracts were immunoblotted for IKBa and p-IKBa. j, k WT and miR-21 KO BMDMs were left unstimulated (control) or stimulated with LPS (500 ng/ml), ATP (5 mM), or nigericin (20 mM) alone or stimulated with LPS (500 ng/ml) for 4 h and then treated with 5 mM ATP or 20 mM nigericin for 30 min. Culture supernatants were analyzed for IL-6 secretion (j) and TNF secretion (k). The data represent the mean ± SD of one among three biological replicates, with three technical replicates each (*P < 0.05; **P < 0.01; Student’s t test)

Fig 4: Both Autophagy and Inflammasome Assembly Defect Triggered by IRGM Leads to Inhibition of NLRP3 Inflammasome Activation(A and B) Analysis of regulation of autophagy flux in control and IRGM knockdown THP-1 cells stimulated with LPS (1 µg/mL, 3 hr) or (A) LPS +ATP (5 mM, 60 min) or (B) LPS+nigericin (5 µM, 15 min) with or without Bafilomycin A1 (300 nM, 3 hr).(C and D) Western blotting analysis of the DSS cross-linked insoluble and soluble fraction of HEK293T cells expressing (C) FLAG-ASC or (D) FLAG-NLRP3 and GFP or GFP-IRGM in absence or presence of chloroquine (50 µM, 5 hr).(E and F) CoIP analysis of interaction between (E) myc-ASC and HA-ASC or (F) GFP-NLRP3 and FLAG-NLRP3 in the absence and presence of GFP-IRGM in HEK293T cells treated or untreated with chloroquine (50 µM, 5 hr).(G–J) Average number of (G and I) ASC specks or (H and J) secreted IL-1 ß from THP-1 GFP-ASC stable cells transfected with control and IRGM siRNA and stimulated with LPS+nigericin, in the absence and presence of (G and H) 3-MA (10 mM, 3 hr) or (I and J) p62 siRNA (n = 3, mean ± SD, *p = 0.05, **p = 0.005, ***p = 0.0005, Student’s unpaired t test).(K–M) HEK293T cells transfected with NLRP3, HA-ASC, caspase-1, pro-IL-1ß, GFP-IRGM, and GFP for 30 hr and (K and M) treated with chloroquine for next 6 hr or (L) transfected with ATG5 siRNA. (K and L) The cell lysates were subjected to western blotting and (M) supernatant used in ELISA to measure IL-1ß. (n = 3, mean ± SD, *p = 0.05, **p = 0.005, Student’s unpaired t test).S.E., short exposure; L.E., long exposure. See also Figures S4 and S5.

Fig 5: Suppression of ATP decreases the pro-inflammatory effect of miRNA-20b in cerebral ischemia. (A) ATP levels were determined using ELISA; densitometric analysis of (B) NLRP3, (C) caspase-1, (D) IL-18 and (E) IL-1ß protein and (F) western blot analysis of NLRP3, caspase-1, IL-18 and IL-1 protein expression following miRNA-20b overexpression and ATP inhibition. Serum levels of (G) IL-1ß and (H) IL-18 following miRNA-20b overexpression and ATP inhibition. **P<0.01 vs. negative control group. ##P<0.01 vs. miRNA-20b overexpression group. miRNA, microRNA; ATP, adenosine 5'-triphosphate; NLRP3, NOD-like receptor pyrin domain containing 3; IL, interleukin; control, negative control group; miRNA-20b, miRNA-20b overexpression group; ATP inhibitor, 10 µM INF39 and miRNA-20b overexpression group.