Journal: Experimental and Therapeutic Medicine

Article Title: CMPK2 accelerates liver ischemia/reperfusion injury via the NLRP3 signaling pathway

doi: 10.3892/etm.2021.10793

Figure Lengend Snippet: Knockdown of CMPK2 decreased the NLRP3-associated inflammation factor. (A) Protein detection of CMPK2 after H/R treatment in scramble siRNA or CMPK2 siRNA-transfected RAW 264.7 cells. (B) Western blotting and subsequent quantification of (C) CMPK2, (D) NLRP3, (E) AIM2, (F) pro-IL-1β, (G) cleaved-caspase-1 (H) IL-1β and (I) IL-18 protein levels. (J) IL-18 and IL-1β levels in the supernatant were detected by ELISA. ** P<0.01, *** P<0.001 vs. control group, # P<0.05, && P<0.01 vs. H/R group. CMPK2, cytidine monophosphate kinase 2; NLRP3, NLR family pyrin domain containing 3; H/R, hypoxia/reoxygenation; siRNA, small interfering RNA; AIM2, absent in melanoma 2.

Article Snippet: Subsequently, the membranes were co-incubated overnight at 4˚C with CMPK2 (cat. no. ab139720; 1:1,000; Abcam), NLRP3 (cat. no. 15101; 1:1,000; Cell Signaling Technology, Inc.), cleaved-caspase-1 (cat. no. sc-398715; 1:1,000; Santa Cruz Biotechnology, Inc.), IL-1β (cat. no. A16288; 1:1,000; ABclonal Biotech Co., Ltd.), IL-18 (cat. no. bs-0529R; 1:1,000; BIOSS), AIM2 (cat. no. sc-515642; 1:1,000, Santa Cruz Biotechnology, Inc.) and β-actin (cat. no. 66009-1-lg; 1:5,000, ProteinTech Group, Inc.).

Techniques: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Small Interfering RNA

Journal: Experimental and Therapeutic Medicine

Article Title: CMPK2 accelerates liver ischemia/reperfusion injury via the NLRP3 signaling pathway

doi: 10.3892/etm.2021.10793

Figure Lengend Snippet: Knockdown of CMPK2 decreases IL-18 and IL-1β by inhibiting the NLRP3 inflammasome, and not AIM2. Protein detection of (A) NLRP3 or (B) AIM2 following H/R treatment in scramble siRNA or specific siRNA-transfected RAW 264.7 cells. (C) Protein detection and respective quantification of (D) cleaved-caspase-1, (E) IL-18 and (F) IL-1β in the H/R + CMPK2 siRNA, H/R + CMPK2 siRNA + NLRP3 siRNA, and H/R + CMPK2 siRNA + AIM2 siRNA groups. ## P<0.01 vs. H/R +CMPK2 siRNA group. CMPK2, cytidine monophosphate kinase 2; NLRP3, NLR family pyrin domain containing 3; H/R, hypoxia/reoxygenation; AIM2, absent in melanoma 2; siRNA, small interfering RNA.

Article Snippet: Subsequently, the membranes were co-incubated overnight at 4˚C with CMPK2 (cat. no. ab139720; 1:1,000; Abcam), NLRP3 (cat. no. 15101; 1:1,000; Cell Signaling Technology, Inc.), cleaved-caspase-1 (cat. no. sc-398715; 1:1,000; Santa Cruz Biotechnology, Inc.), IL-1β (cat. no. A16288; 1:1,000; ABclonal Biotech Co., Ltd.), IL-18 (cat. no. bs-0529R; 1:1,000; BIOSS), AIM2 (cat. no. sc-515642; 1:1,000, Santa Cruz Biotechnology, Inc.) and β-actin (cat. no. 66009-1-lg; 1:5,000, ProteinTech Group, Inc.).

Techniques: Transfection, Small Interfering RNA

Journal: Oncoimmunology

Article Title: AIM2 inflammasome mediates Arsenic-induced secretion of IL-1 β and IL-18

doi: 10.1080/2162402X.2016.1160182

Figure Lengend Snippet: AIM2 deficiency inhibits arsenic-induced cleavage of pro-casepase-1 and secretion of IL-1β and IL-18 in the skin of AIM2 KO mice. (A) AIM2 deficiency was confirmed by protein gel blot (upper panel) and arsenic-induced cleavage of pro-casepase-1 was inhibited in the skin of AIM2 KO mice. (B) Arsenic-induced secretion of IL-1β and IL-18, but not IL-1α, in the skin of wildtype mice was inhibited in AIM2 KO mice. The experiments were repeated three times. Data are represented as mean ± SEM of three experiments. * p < 0.05 vs. control.

Article Snippet: Human AIM2 shRNA plasmid (TR314876), human PKR siRNA (SR303767) and control siRNA (SR30004) was purchased from Origene.

Techniques: Western Blot, Control

Journal: Oncoimmunology

Article Title: AIM2 inflammasome mediates Arsenic-induced secretion of IL-1 β and IL-18

doi: 10.1080/2162402X.2016.1160182

Figure Lengend Snippet: PKR mutation inhibits arsenic-induced activation of AIM2 inflammasome and secretion of IL-1β and IL-18, but not IL-1α, in the skin of C-PKR −/− mice. The C-PKR −/− mice and controlled C57BL/6J mice were treated with 0.25 µM sodium arsenite in drinking water for 8 weeks. The skin tissues were collected. (A) The protein levels of AIM2, cleaved caspase-1, IL-1α, IL-1β and IL-18 were determined by Western-blot and quantified by densitometry. (B) The secretion levels of IL-1α, IL-1β and IL-18 were determined by ELISA assay. The experiments were repeated three times. Data are represented as mean ± SEM of three experiments. * p < 0.05.

Article Snippet: Human AIM2 shRNA plasmid (TR314876), human PKR siRNA (SR303767) and control siRNA (SR30004) was purchased from Origene.

Techniques: Mutagenesis, Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Nature metabolism

Article Title: CRAT links cholesterol metabolism to innate immune responses in the heart.

doi: 10.1038/s42255-023-00844-5

Figure Lengend Snippet: Fig. 5 | Depletion of CRAT activates DNA-sensing AIM2 inflammasome. NRVMs were transduced with lentivirus expressing control shRNA or Crat shRNA for 5 d. All transduced cells expressed green fluorescent protein (GFP). a, Cells were fixed for immunostaining with anti-AIM2 antibody. Representative images showed that knockdown of CRAT induced the formation of AIM2 specks. Scale bar, 25 μm. b–d, Western blots and quantification analyses indicated that the cleavage of pro-caspase-1 (c) and pro-IL-1β (d) was increased in CRAT-deficient NRVMs. e, Representative images of NRVMs stained with cardiac troponin-T antibody

Article Snippet: Lentiviral vectors encoding Crat, cGas, Pparα, Aim2 shRNAs or control vectors were obtained from Origene.

Techniques: Transduction, Expressing, Control, shRNA, Immunostaining, Knockdown, Western Blot, Staining

Journal: Nature metabolism

Article Title: CRAT links cholesterol metabolism to innate immune responses in the heart.

doi: 10.1038/s42255-023-00844-5

Figure Lengend Snippet: Fig. 7 | Schematic model of the role of CRAT in cholesterol metabolism and innate immune responses. Loss of CRAT promotes cholesterol catabolism through the bile acid synthesis pathway, leading to intracellular accumulation of intermediates of bile acid synthesis such as 7-HOCA, which is sufficient to promote mtDNA release into cytosol and trigger cGAS–STING-dependent type I interferon responses. Further, type I interferon responses elicited by CRAT deficiency lead to a substantial increase in AIM2 expression and subsequent activation of the DNA-sensing AIM2 inflammasome, which in turn promotes proteolytic maturation of IL-1β and CM pyroptosis. Eventually, genetic deletion of CRAT in CMs leads to myocardial inflammation and results in DCM. Collectively, we have identified a mechanism by which cardiac energy metabolism, cholesterol homeostasis and CM-intrinsic innate immune responses are interconnected via a CRAT-mediated bile acid synthesis pathway, which contributes to chronic myocardial inflammation and HF progression.

Article Snippet: Lentiviral vectors encoding Crat, cGas, Pparα, Aim2 shRNAs or control vectors were obtained from Origene.

Techniques: Expressing, Activation Assay