il 1β elisa  (R&D Systems)

Journal: International journal of molecular sciences

Article Title: Vitamin A Promotes the Fusion of Autophagolysosomes and Prevents Excessive Inflammasome Activation in Dextran Sulfate Sodium-Induced Colitis.

doi: 10.3390/ijms24108684

Figure Lengend Snippet: Figure 1. VAD B6 mice show more severe DSS-induced colitis than VAS mice. (a–d) VAS and VAD B6 mice were fed 4% DSS and were followed up until day 10. (a) Macroscopic findings (n = 3), (b) survival rate (n = 12), (c) body weight course (n = 8), (d) colonic length (n = 6), (e) H&E staining, and (f) histological scoring (n = 4) are shown. In (b), data were analyzed using the Kaplan–Meier test. In (c,d,f), data are presented as mean ± SEM (n > 2 independent experiments). An unpaired t-test was used for statistical analysis (* p < 0.05, ** p < 0.01). DSS, dextran sulfate sodium; H&E, hematoxylin and eosin; SEM, standard error of the mean; VAD, vitamin A-deficient; VAS, vitamin A-sufficient.

Article Snippet: Protein expression was detected by Western blotting with anti-caspase-11 primary antibody (1:1000) (ab 180673; Abcam), anti-GSDMD primary antibody (1:100) (ab 209845; Abcam), anti-NLRP3 primary antibody (1:1000) (AG-20B-0014; AdipoGen, Basel, Switzerland), anti-ASC primary antibody (1:1000) (sc-22514; Santa Cruz), anti-caspase-1 primary antibody (1:500) (sc-514; Santa Cruz), anti-IL-1β primary antibody (1:1000) (MAB401; R&D systems Inc., Minneapolis, MN, USA), anti-α-tubulin antibody (1:1000) (21255; Cell Signaling), and horseradish peroxidaseconjugated secondary antibody (1:5000–10,000) (R&D systems Inc., Minneapolis, MN, USA).

Techniques: Staining

Journal: International journal of molecular sciences

Article Title: Vitamin A Promotes the Fusion of Autophagolysosomes and Prevents Excessive Inflammasome Activation in Dextran Sulfate Sodium-Induced Colitis.

doi: 10.3390/ijms24108684

Figure Lengend Snippet: Figure 3. Inflammasome-related proteins increase in colonic lamina propria of VAD mice. (a–c) Profile of cytokine production in lamina propria (LP) from DSS-treated VAD mice using a multiplex system (Bio-Plex, Bio-Rad) (n = 5). VAS and VAD mice were fed with DSS in drinking water as described in the Materials and Methods. On day 0, 1, and 2 after DSS feeding, colonic LP of mice was isolated using a modified Bjerk’s method. The cytokine assay was performed using Bio-Plex multi-assay system (Bio-Rad Laboratories). (d) Immunoblotting for inflammasome-related proteins was performed using LP isolated from DSS-treated mice. (e) Caspase-1 activities in colonic LP were measured using a colorimetric protease assay kit (Bio Vision Research Product) (n = 5). Data with error bars are represented as means ± SEM (n > 2 independent experiments). An unpaired t-test was used for statistical analysis (NS, not significant; * p < 0.05, ** p < 0.01). Each panel represents an experiment performed at least three times. DSS, dextran sulfate sodium; SEM, standard error of the mean; VAD, vitamin A-deficient.

Article Snippet: Protein expression was detected by Western blotting with anti-caspase-11 primary antibody (1:1000) (ab 180673; Abcam), anti-GSDMD primary antibody (1:100) (ab 209845; Abcam), anti-NLRP3 primary antibody (1:1000) (AG-20B-0014; AdipoGen, Basel, Switzerland), anti-ASC primary antibody (1:1000) (sc-22514; Santa Cruz), anti-caspase-1 primary antibody (1:500) (sc-514; Santa Cruz), anti-IL-1β primary antibody (1:1000) (MAB401; R&D systems Inc., Minneapolis, MN, USA), anti-α-tubulin antibody (1:1000) (21255; Cell Signaling), and horseradish peroxidaseconjugated secondary antibody (1:5000–10,000) (R&D systems Inc., Minneapolis, MN, USA).

Techniques: Multiplex Assay, Isolation, Cytokine Assay, Western Blot, Protease Assay

Journal: International journal of molecular sciences

Article Title: Vitamin A Promotes the Fusion of Autophagolysosomes and Prevents Excessive Inflammasome Activation in Dextran Sulfate Sodium-Induced Colitis.

doi: 10.3390/ijms24108684

Figure Lengend Snippet: Figure 4. Macrophages pretreated with RAR antagonist exhibit increased pyroptosis mediated via non-canonical inflammasome signaling. (a–m) Effect of RAR antagonist (Ro41-5253) on cell viability of murine macrophage cells (RAW 264.7) treated with LPS. RAW 264.7 cells were cultured in 10 mg/mL LPS for 24 h, pretreated with or without 10 mM Ro41-5253 for 1 h. Cell viability, cytotoxicity, cytokine production, and immunoblotting were examined at each time point. (a) Cell viability in the absence of LPS (n = 4), (b) cell viability in the presence of 10 mg/mL LPS (n = 4), (c) representative images of supernatants, and (d–g) morphology of RAW 264.7 cells upon Ro41-5253 and LPS treatment. (h,i) LDH was measured using Cytotox96 (non-radioactive cytotoxicity assay, Promega). (h) Cytotoxicity assay without LPS (n = 6) and (i) cytotoxicity assay in the presence of 10 mg/mL LPS (n = 6). (j–l) Titers of IL-1β, IL-18, and TNF-α in supernatants were measured using ELISA. (j) IL-1β production (n = 4), (k) IL-18 production (n = 4), (l) TNF-α production (n = 4), and (m) immunoblotting of RAW 264.7 cells upon Ro41-5253 and LPS treatment. Graphs show the mean ± SEM of duplicate wells and represent three independent experiments. An unpaired t-test was used for statistical analysis (NS, not significant; * p < 0.05, ** p < 0.01). Each panel represents an experiment performed at least three times. IL-1β, -18, interleukin-1β, -18; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; RAR, retinoic acid receptor; TNF-α, tumor necrosis factor-α.

Article Snippet: Protein expression was detected by Western blotting with anti-caspase-11 primary antibody (1:1000) (ab 180673; Abcam), anti-GSDMD primary antibody (1:100) (ab 209845; Abcam), anti-NLRP3 primary antibody (1:1000) (AG-20B-0014; AdipoGen, Basel, Switzerland), anti-ASC primary antibody (1:1000) (sc-22514; Santa Cruz), anti-caspase-1 primary antibody (1:500) (sc-514; Santa Cruz), anti-IL-1β primary antibody (1:1000) (MAB401; R&D systems Inc., Minneapolis, MN, USA), anti-α-tubulin antibody (1:1000) (21255; Cell Signaling), and horseradish peroxidaseconjugated secondary antibody (1:5000–10,000) (R&D systems Inc., Minneapolis, MN, USA).

Techniques: Cell Culture, Western Blot, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay

Journal: International journal of molecular sciences

Article Title: Vitamin A Promotes the Fusion of Autophagolysosomes and Prevents Excessive Inflammasome Activation in Dextran Sulfate Sodium-Induced Colitis.

doi: 10.3390/ijms24108684

Figure Lengend Snippet: Figure 5. VAD decreases autophagic flux by reducing the fusion of autophagolysosomes. (a) Im- munoblotting for autophagy-related proteins was performed using isolated colonic lamina propria (LP) of B6 mice (day 2). (b) Autophagy in colonic tissues of GFP-LC3 transgenic mice (GFP-LC3#53) (day 1). (c) Electron microscopic analysis of colonic LP with or without DSS treatment (day 1). The areas of red box were magnified. The red arrows indicate mitochondria. (d,e) Effects of RAR antagonist (Ro41-5253) on autophagy of murine macrophage cells (RAW 264.7). (d) Immunoblotting of autophagy-related proteins. RAW 264.7 cells were pretreated with or without 10 mM Ro41-5253 for 1 h and then harvested in the presence of 10 mg/mL LPS after 24 h. (e) Mitochondrial ROS was measured by MitoSOX staining after 4 h of EBSS treatment. Each panel represents an experiment performed at least three times. EBBSS, Earle’s Balanced Salt Solution; LPS, lipopolysaccharide; RAR, retinoic acid receptor; ROS, reactive oxygen species. #### indicates a value greater than 9999.

Article Snippet: Protein expression was detected by Western blotting with anti-caspase-11 primary antibody (1:1000) (ab 180673; Abcam), anti-GSDMD primary antibody (1:100) (ab 209845; Abcam), anti-NLRP3 primary antibody (1:1000) (AG-20B-0014; AdipoGen, Basel, Switzerland), anti-ASC primary antibody (1:1000) (sc-22514; Santa Cruz), anti-caspase-1 primary antibody (1:500) (sc-514; Santa Cruz), anti-IL-1β primary antibody (1:1000) (MAB401; R&D systems Inc., Minneapolis, MN, USA), anti-α-tubulin antibody (1:1000) (21255; Cell Signaling), and horseradish peroxidaseconjugated secondary antibody (1:5000–10,000) (R&D systems Inc., Minneapolis, MN, USA).

Techniques: Isolation, Transgenic Assay, Western Blot, Staining

Journal: International journal of molecular sciences

Article Title: Vitamin A Promotes the Fusion of Autophagolysosomes and Prevents Excessive Inflammasome Activation in Dextran Sulfate Sodium-Induced Colitis.

doi: 10.3390/ijms24108684

Figure Lengend Snippet: Figure 6. VAD impairs autophagic and listericidal activities of macrophages and host resistance to Listeria monocytogenes infection. (a–c) C57BL/6J and SCID mice were intravenously infected with 5 × 105 CFU of L. monocytogenes. Mice were followed up until day 10, or spleens and livers were harvested 24 h (day 1) and 48 h (day 2) post-infection. (a) Survival rate (n = 12), (b) the number of bacteria in organs (day 1, n = 4), (c) the number of bacteria in organs (day 2, n = 4), and (d) splenic macrophages of VAS and VAD mice infected with L. monocytogenes at the multiplicity of infection (MOI) of 10. The numbers of viable bacteria in cell lysates were counted at each time point (n = 5). (e) Fluorescence microscopic findings of GFP-LC3 transgenic mice (GFP-LC3#53) infected with DsRedEx-labeled L. monocytogenes. Mice were intravenously infected with 5 × 105 CFU of L. monocytogenes. The livers were removed 48 h (day 2) post-infection. After fixation by perfusion with 4% paraformaldehyde (PFA), fluorescent signals were observed under a fluorescence microscope. (f) Electron microscopic findings of mice infected with L. monocytogenes. Mice were intravenously infected with 5 × 105 CFU of L. monocytogenes. The spleens were harvested pre-infection (day 0) and 24 h (day 1) post-infection. The red circles indicate autophagosomes. In (a), data were analyzed using the Kaplan–Meier test. In (b–d), data are presented as mean ± SEM (n > 2 independent experiments). An unpaired t-test was used for statistical analysis (NS, not significant; * p < 0.05, ** p < 0.01). Each panel represents an experiment performed at least three times. CFU, colony-forming unit; SEM, standard error of the mean; VAD, vitamin A-deficient; VAS, vitamin A-sufficient.

Article Snippet: Protein expression was detected by Western blotting with anti-caspase-11 primary antibody (1:1000) (ab 180673; Abcam), anti-GSDMD primary antibody (1:100) (ab 209845; Abcam), anti-NLRP3 primary antibody (1:1000) (AG-20B-0014; AdipoGen, Basel, Switzerland), anti-ASC primary antibody (1:1000) (sc-22514; Santa Cruz), anti-caspase-1 primary antibody (1:500) (sc-514; Santa Cruz), anti-IL-1β primary antibody (1:1000) (MAB401; R&D systems Inc., Minneapolis, MN, USA), anti-α-tubulin antibody (1:1000) (21255; Cell Signaling), and horseradish peroxidaseconjugated secondary antibody (1:5000–10,000) (R&D systems Inc., Minneapolis, MN, USA).

Techniques: Infection, Bacteria, Fluorescence, Transgenic Assay, Labeling, Microscopy

Journal: International journal of molecular sciences

Article Title: Vitamin A Promotes the Fusion of Autophagolysosomes and Prevents Excessive Inflammasome Activation in Dextran Sulfate Sodium-Induced Colitis.

doi: 10.3390/ijms24108684

Figure Lengend Snippet: Figure 7. Blockade of IL-1β ameliorates DSS-induced colitis in VAD mice. (a,b) Effect of monoclonal antibody against IL-1β on DSS-induced colitis in VAD B6 mice. VAD B6 mice were injected intraperi- toneally with 50 mg/head/day of monoclonal antibody against IL-1β (BioLegend) from day 0 (when DSS treatment was initiated) to day 3. Isotype-matched IgG was injected as a control. (a) Survival rate, (b) body weight course. (c,d) Survival rate and body weight course after MCC950 administration. (e,f) Survival rate and body weight course after rapamycin administration. In (a,c,e), data were analyzed using the Kaplan–Meier test. In (b), data are presented as mean ± SEM (n > 2 independent experiments). An unpaired t-test test was used for statistical analysis (NS, not significant; ** p < 0.01). DSS, dextran sulfate sodium; IL-1β, interleukin-1β; SEM, standard error of the mean; VAD, vitamin A-deficient.

Article Snippet: Protein expression was detected by Western blotting with anti-caspase-11 primary antibody (1:1000) (ab 180673; Abcam), anti-GSDMD primary antibody (1:100) (ab 209845; Abcam), anti-NLRP3 primary antibody (1:1000) (AG-20B-0014; AdipoGen, Basel, Switzerland), anti-ASC primary antibody (1:1000) (sc-22514; Santa Cruz), anti-caspase-1 primary antibody (1:500) (sc-514; Santa Cruz), anti-IL-1β primary antibody (1:1000) (MAB401; R&D systems Inc., Minneapolis, MN, USA), anti-α-tubulin antibody (1:1000) (21255; Cell Signaling), and horseradish peroxidaseconjugated secondary antibody (1:5000–10,000) (R&D systems Inc., Minneapolis, MN, USA).

Techniques: Injection, Control