Journal: Gut Microbes

Article Title: An IBD-associated pathobiont synergises with NSAID to promote colitis which is blocked by NLRP3 inflammasome and Caspase-8 inhibitors

doi: 10.1080/19490976.2022.2163838

Figure Lengend Snippet: Reagent and resources.

Article Snippet: Rabbit monoclonal cleaved gasdermin D (GSDMD) , Cell signaling technology , Cat# 10137S.

Techniques: Blocking Assay, Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Biomarker Assay, Sequencing, Software

Journal: Frontiers in Immunology

Article Title: Complement membrane attack complex is an immunometabolic regulator of NLRP3 activation and IL-18 secretion in human macrophages

doi: 10.3389/fimmu.2022.918551

Figure Lengend Snippet: Reagents List.

Article Snippet: Rabbit anti-Gasdermin D L60 , Cell Signalling , 93709.

Techniques: Purification, Recombinant, In Vivo, Generated, Enzyme-linked Immunosorbent Assay, Sequencing, Software

Journal: Cell Communication and Signaling : CCS

Article Title: Mesenchymal stem cells protect against TBI-induced pyroptosis in vivo and in vitro through TSG-6

doi: 10.1186/s12964-022-00931-2

Figure Lengend Snippet: Pyroptosis in the murine TBI model. To assess the expression of pyroptosis-related proteins, TBI mice were sacrificed and brain tissues were removed at 12 h, 24 h, 48 h and 72 h post-TBI surgery. a Representative protein bands and corresponding grayscale values of NLRP3, pro-Caspase-1, Caspase-1 p20, GSDMD, and cleaved-GSDMD performed by western blotting. b – d Representative double immunofluorescence staining photographs and quantification of Iba-1 (green) with NLRP3 (red), Caspase-1 p20 (red), or GSDMD (red) in the injured cortex. Scale bar = 50 μm. All data are represented as means ± SD (n = 5 mice/group) and compared by t -test. *p < 0.05, ** p < 0.01, *** p < 0.001 versus sham group

Article Snippet: The membranes were blocked for 2 h at 37 °C with 5% non-fat milk and 0.05% Tween-20, then incubated overnight at 4 °C with Mouse anti-GAPDH (60004-1-Ig, Proteintech, 1:5000), rabbit anti-NLRP3 (#15101, CST, 1:1000), rabbit anti-pro-Caspase-1 (AF5418, Affinity, 1:1000), rabbit anti-Caspase-1 p20 (AF4005, Affinity, 1:1000), rabbit anti-GSDMD (ab219800, Abcam, 1:1000), and rabbit anti-cleaved-GSDMD (#10137, CST, 1:1000).

Techniques: Expressing, Western Blot, Double Immunofluorescence Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Mesenchymal stem cells protect against TBI-induced pyroptosis in vivo and in vitro through TSG-6

doi: 10.1186/s12964-022-00931-2

Figure Lengend Snippet: hUMSCs downregulated the NLRP3/Caspase-1/GSDMD signaling pathway and reduced microglia pyroptosis in TBI mice via TSG-6. TBI mice were intracerebroventricularly injected with 10 μL of PBS, 0.5 µg rmTSG-6 or 1 µg rmTSG-6, or 2.5 × 10 5 hUMSCs, sh-TSG-6 hUMSCs, sh-NC hUMSCs, in 10 μL of PBS at 4 h after TBI surgery. Then TBI mice were sacrificed and brain tissues were removed at 48 h post-TBI surgery. a Representative protein bands and corresponding grayscale values of NLRP3, pro-Caspase-1, Caspase-1 p20, GSDMD, and cleaved-GSDMD performed by western blotting. b – d Representative double immunofluorescence staining photographs and quantification of Iba-1 (green) with NLRP3 (red), Caspase-1 p20 (red), or GSDMD (red) in the injured cortex. Scale bar = 50 μm. All data are represented as means ± SD (n = 6 mice/group) and compared by one-way ANOVA, followed by the Tukey’s post hoc test. * p < 0.05, ** p < 0.01, versus TBI group; # p < 0.05, sh-TSG-6 hUMSC group versus sh-NC hUMSC group

Article Snippet: The membranes were blocked for 2 h at 37 °C with 5% non-fat milk and 0.05% Tween-20, then incubated overnight at 4 °C with Mouse anti-GAPDH (60004-1-Ig, Proteintech, 1:5000), rabbit anti-NLRP3 (#15101, CST, 1:1000), rabbit anti-pro-Caspase-1 (AF5418, Affinity, 1:1000), rabbit anti-Caspase-1 p20 (AF4005, Affinity, 1:1000), rabbit anti-GSDMD (ab219800, Abcam, 1:1000), and rabbit anti-cleaved-GSDMD (#10137, CST, 1:1000).

Techniques: Injection, Western Blot, Double Immunofluorescence Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Mesenchymal stem cells protect against TBI-induced pyroptosis in vivo and in vitro through TSG-6

doi: 10.1186/s12964-022-00931-2

Figure Lengend Snippet: hUMSCs reduced LPS + ATP-induced BV2 microglia pyroptosis by secreting TSG-6. BV2 cells were co-cultured with hUMSCs, sh-TSG-6 hUMSCs, or sh-NC hUMSCs activated by TNF-α and different concentration of rmTSG-6 (50 ng/mL, 100 ng/mL, 200 ng/mL) and treated with or without LPS (1 µg/mL) + ATP (5 mM) for 12 h. a Representative photomicrographs and quantification of double staining with PI (red) and DAPI (blue) were captured by confocal microscopy. b Caspase-1 activity and the release of LDH in the supernatant was assessed using Caspase-1 activity and LDH activity detection kits. c The protein levels of IL-1β, pro IL-18, and TNF-α were evaluated by ELISA. d Representative protein bands and corresponding grayscale values of NLRP3, pro-Caspase-1, Caspase-1 p20, GSDMD and cleaved-GSDMD were performed by western blotting. Scale bar = 100 μm. All data are represented as means ± SD of at least 3 independent experiments and compared by one-way ANOVA, followed by the Tukey’s post hoc test. * p < 0.05, ** p < 0.01, versus LPS + ATP group; # p < 0.05, sh-TSG-6 hUMSC group versus sh-NC hUMSC group

Article Snippet: The membranes were blocked for 2 h at 37 °C with 5% non-fat milk and 0.05% Tween-20, then incubated overnight at 4 °C with Mouse anti-GAPDH (60004-1-Ig, Proteintech, 1:5000), rabbit anti-NLRP3 (#15101, CST, 1:1000), rabbit anti-pro-Caspase-1 (AF5418, Affinity, 1:1000), rabbit anti-Caspase-1 p20 (AF4005, Affinity, 1:1000), rabbit anti-GSDMD (ab219800, Abcam, 1:1000), and rabbit anti-cleaved-GSDMD (#10137, CST, 1:1000).

Techniques: Cell Culture, Concentration Assay, Double Staining, Confocal Microscopy, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Cigarette smoke-induced gasdermin D activation in bronchoalveolar macrophages and bronchial epithelial cells dependently on NLRP3

doi: 10.3389/fimmu.2022.918507

Figure Lengend Snippet: Gasdermin d -deficient mice ( Gsdmd -/- ) have reduced pulmonary inflammation and remodeling after acute smoke cigarette (CS) exposure. WT and Gsdmd -/- mice were exposed to CS or Air during 4 days. Total cells (A) , macrophages (B) and neutrophils (C) counts and MPO level in BAL (D) are shown. CXCL1 (E) , CXCL5 (F) , CXCL15 (G) and IL-1β (H) levels were measured in BAL. MMP-9 (I) and TIMP-1 (J) remodeling factor levels were measured in BAL. In lungs, levels of IL-1β (K) , MMP-9 (L) and TIMP-1 (M) were also measured. All parameters decreased in Gsdmd -/- mice exposed to CS as compared to CS WT mice. Data are representative of four experiments and are expressed as mean values ± SEM (n= 4-6 mice per group, *p < 0.05, **p < 0.01, ns, non significant, using a Mann Whitney test).

Article Snippet: Cells were washed 3 times in TBS, incubated 15 min in TBS-0.3% Triton X-100, then washed 3 times in TBS, blocked in TBS-10% Bovine Serum Albumin (BSA) for 45min and incubated overnight with primary rabbit anti-Cleaved Caspase1 (1:100, #89332, Cell Signaling) or anti-Cleaved Gasdermin (1:100, #10137, Cell Signaling).

Techniques: MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Cigarette smoke-induced gasdermin D activation in bronchoalveolar macrophages and bronchial epithelial cells dependently on NLRP3

doi: 10.3389/fimmu.2022.918507

Figure Lengend Snippet: Gasdermin d -deficient mice ( Gsdmd -/- ) have reduced pulmonary inflammation and remodeling after acute smoke cigarette (CS) exposure. WT and Gsdmd -/- mice were exposed to CS or Air during 4 days. Total cells (A) , macrophages (B) and neutrophils (C) counts and MPO level in BAL (D) are shown. CXCL1 (E) , CXCL5 (F) , CXCL15 (G) and IL-1β (H) levels were measured in BAL. MMP-9 (I) and TIMP-1 (J) remodeling factor levels were measured in BAL. In lungs, levels of IL-1β (K) , MMP-9 (L) and TIMP-1 (M) were also measured. All parameters decreased in Gsdmd -/- mice exposed to CS as compared to CS WT mice. Data are representative of four experiments and are expressed as mean values ± SEM (n= 4-6 mice per group, *p < 0.05, **p < 0.01, ns, non significant, using a Mann Whitney test).

Article Snippet: Lung sections were permeabilized in PBS 0.5% triton X-100, blocked with 5% FCS for 1h at RT and then incubated overnight with primary mouse anti-cleaved Gasdermin D (1:100, #10137, Cell Signaling).

Techniques: MANN-WHITNEY