Journal: bioRxiv

Article Title: Pyruvate dehydrogenase kinase supports macrophage NLRP3 inflammasome activation during acute inflammation

doi: 10.1101/2021.10.02.462869

Figure Lengend Snippet: BMDMs or thioglycollate-elicited peritoneal macrophages (PMs) were stimulated with LPS (300 ng/ml) for 3 h and ATP for 1 h to induce the NLRP3 inflammasome activation. DCA (20 mM) or JX06 (10 μM) were added after LPS priming. ( A ) The targeting sites of the pharmacological inhibitors are indicated. ( B-F ) IL-1β secretion from LPS plus ATP-treated BMDMs. Hexokinase inhibitor 2-deoxy-D-glucose (2-DG; 10 mM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibitor heptelidic acid (HA, 15 μM), lactate dehydrogenase inhibitor sodium oxamate (40 mM), glucose-6-phosphate dehydrogenase (G6PD) inhibitor dehydroepiandrosterone (DHEA) (200 μM), mitochondrial pyruvate carrier (MPC) inhibitor UK5099 (5 μM), pyruvate carboxylase (PC) inhibitor chlorothricin (100 μM), or pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGDH) inhibitor CPI-613 (200 μM), were added 30 min before ATP. ( G ) Pdhe1a mRNA expression in control and PDHe1a siRNA transfected elicited PMs. ( H-I ) IL-1β secretion (H) and caspase-1 cleavage (I) in the culture supernatant from control siRNA and Pdhe1a siRNA transfected elicited PMs treated with or without LPS plus ATP in the presence or absence of DCA or JX06. ( J ) mRNA expression of PDHK isoforms in BMDMs treated with or without LPS plus ATP. Gene expression was normalized to PDHK1 expression in unstimulated macrophages. ( K ) Transcript expression of PDHK isoform 1, 2, or 4 in control and isoform-specific siRNA transfected elicited PMs, respectively. ( L-M ) IL-1β secretion (L) and caspase-1 cleavage (M) in the culture supernatant from control siRNA and isoform-specific siRNA transfected elicited PMs treated with or without LPS plus ATP. Groups with different letters are significantly different (p<0.05); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: non-significant. One-way ANOVA with post hoc Tukey’s multiple comparisons test ; unpaired, two-tailed student’s T-test ( , and ).

Article Snippet: In some experiments, LPS-primed macrophages were treated with 2-DG (10 mM, Sigma-Aldrich), heptelidic acid (15 μM, Cayman Chemical), sodium oxamate (40 mM, Santa Cruz Biotechnology), DHEA (200 μM, EMD Millipore), UK5099 (5 μM, Cayman Chemical), CPI-613 (200 μM, Cayman Chemical), chlorothricin (100 μM, Cayman Chemical), AOAA (1 mM, Cayman Chemical), fumonisin B1 (15 μM, Cayman Chemical), MYLS22 (50 μM, MedChemExpress), ML385 (5 μM, MedChemExpress), etomoxir (50 μM, Cayman Chemical), bafilomycin A1 (50 nM, In vivoGen), or 3-MA (5 mM, Sigma-Aldrich), as indicated in the figure legends.

Techniques: Activation Assay, Expressing, Transfection, Two Tailed Test

Journal: Cellular and Molecular Immunology

Article Title: NLRP3-dependent pyroptosis is required for HIV-1 gp120-induced neuropathology

doi: 10.1038/s41423-019-0260-y

Figure Lengend Snippet: The CXCR4-Kv1.3 axis is implicated in gp120 LAV-induced NLRP3 inflammasome activation. a–f ELISA of IL-1β in supernatants of BV2 cells stimulated by gp120 LAV (0.5 μg/ml) in the presence of increasing doses of KCl (50–200 mM), the K+ efflux inhibitor glyburide (50–200 μM) (a); the ROS inhibitor ADPC (5–50 μM), NAC (5–25 μM) (b); MitoTEMPO (100–500 μM) (c); the actin polymerization inhibitor cytochalasin D (1–25 μM) (d); the lysosome inhibitor bafilomycin A (10–200 nM) (e); and the cathepsin B inhibitor CA-074Me (5–20 μM) (f). g Western blot (upper panel) and densitometric analysis (lower panel) of Kv1.3 in lysates of BV2 cells stimulated with increasing doses of gp120 LAV (0.01–0.5 μg/ml). h, i ELISA of IL-1β in supernatants of BV2 cells stimulated with gp120 LAV (0.5 μg/ml) in the presence of increasing doses of a Kv1.3-specific inhibitor (PAP-1, 0.1–2 μM) (h) or CXCR4-specific inhibitor (AMD3100, 0.1–10 μM) (i). j, k Immunoblot analysis of Casp-1-p20 and IL-1β-p17 (j) and ELISA of IL-1β (k) in supernatants (Sup) of BV2 cells transfected with control siRNA, CXCR4 or Kv1.3-specific siRNA and treated with or without gp120 LAV (0.5 μg/ml). CXCR4, Kv1.3, NLRP3, pro-casp-1, and pro-IL-1β in lysates (Lys) of these cells were also detected. l–n Western blots (l) and densitometric analysis of Kv1.3 (m) and p38-MAPK phosphorylation (P-p38, n) in lysates of BV2 cells transfected with siRNA control or CXCR4-specific siRNA and stimulated with or without gp120 LAV (0.5 μg/ml). The data are displayed as the mean ± SEM (n = 5) from three independent experiments (a–i, k, m, n). The Western blot results are representative of three (j) and five (g, l) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: The primary antibodies used are as follows: rabbit anti-IL-1β (1:2000), rabbit anti-arginase-1 (1:1000), rabbit anti-NF-κB p65 (1:50,000), rabbit anti-RELM alpha (1:3000), rabbit anti-CXCR4 (1:1000), rabbit anti-NLRP1 (1:1000), and rabbit anti-NLRP3 (1:1000) were all purchased from Abcam (Cambridge, USA); rabbit anti-caspase-1 (1:2000), rabbit anti-AIM2 (1:2000), rabbit anti-IκBα (1:2000), rabbit anti-GSDMD (1:2000), rabbit anti-Histone H3 (1:6000), and rabbit anti-Kv1.3 (1:2000) were all purchased from Proteintech Group (Chicago, USA); rabbit anti-β-actin (1:6000) (Bioss Antibodies, USA); mouse anti-ASC (1:200) (Santa Cruz Biotechnology, USA) were used; rabbit anti-NLRC4 (1:800) and rabbit anti-iNOS (1:800) were purchased from Boster Biotechnology (Wuhan, China); rabbit anti-cleaved-caspase-1 (1:1000), rabbit anti-cleaved-IL-1β (1:1000), rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (1:1000), and rabbit anti-p38 MAPK (1:1000) were all purchased from Cell Signaling Technology (Danvers, USA).

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