fbx  (Selleck Chemicals)

Journal: Life Science Alliance

Article Title: Inhibition of xanthine oxidoreductase with febuxostat, but not allopurinol, prevents inflammasome assembly and IL-1β release

doi: 10.26508/lsa.202403191

Figure Lengend Snippet: (B, C, D, E, F, G, H, I) Mouse bone marrow–derived macrophages were pre-treated for 30 min with DMSO vehicle (Veh), Fbx (200 µM), Allo (250 μg/ml), or Casp-i (Z-YVAD-FMK, 20 μM), and then with LPS (100 ng/ml) and IFNγ (20 ng/ml) for 6 h (B, C, D, E, F, G, H, I) or 18 h (H). (E, F, G, H, I) In experiments in which cytokine release was measured (E, F, G, H), ATP (1 mM) was added 3 h before harvesting (E, F, G, H, I). (A) Schematic representation of the macrophage differentiation and polarization protocol. (B, C, D) Tnfa , Il6 , and Il1b gene expression measured by qRT-PCR. (E, F, G) TNF-ɑ, IL-6, and IL-1β protein levels were measured by ELISA. Results are represented as the percentage of M(LPS+γ). (H) pro-IL1β frequency in F4/80 + cells determined by flow cytometry. (I) Caspase-1 activity (relative luminescence units, RLU) was measured according to the manufacturer’s protocol. Results are represented as the percentage of M(LPS+γ). (J) Caspase inhibition screening test (relative fluorescence units, RFU). Caspase-1 inhibitor Ac-YVAD-CHO was used as a control, according to the manufacturer’s instructions. Results are the mean ± SEM of at least five biological replicates. P -values were calculated (one-way ANOVA) on the data before normalization. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Red blood cells were lysed with red blood cell lysis buffer (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.3–7.4) for 3 min, and residual cells were plated in RPMI containing 10% FBS, 1% Pen/Strep, 1% HEPES (#SH3023701; Thermo Fisher Scientific), and 20% L929 media, for 7 d. After differentiation, macrophages were pre-treated with DMSO (# BP231-100; Thermo Fisher Scientific), FBX (200 μM; #S1547; Selleck Chemicals), Allo (250 μg/ml; #A8003; Sigma-Aldrich), or Casp-i (Z-YVAD-FMK, 20 μM; #218746; Sigma-Aldrich) for 30 min, and then with LPS (100 ng/ml; #L2630; Sigma-Aldrich) and IFNγ (20 ng/ml; #315-05; PeproTech), according to figure legends.

Techniques: Derivative Assay, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Activity Assay, Inhibition, Fluorescence, Control

Journal: Life Science Alliance

Article Title: Inhibition of xanthine oxidoreductase with febuxostat, but not allopurinol, prevents inflammasome assembly and IL-1β release

doi: 10.26508/lsa.202403191

Figure Lengend Snippet: Mouse bone marrow–derived macrophages were pre-treated for 30 min with DMSO vehicle (Veh), Fbx (200 µM), Allo (250 μg/ml), or Casp-i (Z-YVAD-FMK, 20 μM), followed by polarization with LPS (100 ng/ml) and IFNγ (20 ng/ml) for 6 h. Frequency of live cells by flow cytometry. Results are representative of at least four biological replicates.

Article Snippet: Red blood cells were lysed with red blood cell lysis buffer (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.3–7.4) for 3 min, and residual cells were plated in RPMI containing 10% FBS, 1% Pen/Strep, 1% HEPES (#SH3023701; Thermo Fisher Scientific), and 20% L929 media, for 7 d. After differentiation, macrophages were pre-treated with DMSO (# BP231-100; Thermo Fisher Scientific), FBX (200 μM; #S1547; Selleck Chemicals), Allo (250 μg/ml; #A8003; Sigma-Aldrich), or Casp-i (Z-YVAD-FMK, 20 μM; #218746; Sigma-Aldrich) for 30 min, and then with LPS (100 ng/ml; #L2630; Sigma-Aldrich) and IFNγ (20 ng/ml; #315-05; PeproTech), according to figure legends.

Techniques: Derivative Assay, Flow Cytometry

Journal: Life Science Alliance

Article Title: Inhibition of xanthine oxidoreductase with febuxostat, but not allopurinol, prevents inflammasome assembly and IL-1β release

doi: 10.26508/lsa.202403191

Figure Lengend Snippet: (A) Gating strategy to identify F4/80 + BMDMs. (B) Gating strategy showing CD86 + , pro-IL-1β + , and iNOS + BMDMs from F4/80 + gate pre-treated for 30 min with DMSO vehicle (Veh), Fbx (200 µM), Allo (250 μg/ml), or Casp-i (Z-YVAD-FMK, 20 μM), and then with LPS (100 ng/ml) and IFNγ (20 ng/ml) for 18 h.

Article Snippet: Red blood cells were lysed with red blood cell lysis buffer (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.3–7.4) for 3 min, and residual cells were plated in RPMI containing 10% FBS, 1% Pen/Strep, 1% HEPES (#SH3023701; Thermo Fisher Scientific), and 20% L929 media, for 7 d. After differentiation, macrophages were pre-treated with DMSO (# BP231-100; Thermo Fisher Scientific), FBX (200 μM; #S1547; Selleck Chemicals), Allo (250 μg/ml; #A8003; Sigma-Aldrich), or Casp-i (Z-YVAD-FMK, 20 μM; #218746; Sigma-Aldrich) for 30 min, and then with LPS (100 ng/ml; #L2630; Sigma-Aldrich) and IFNγ (20 ng/ml; #315-05; PeproTech), according to figure legends.

Techniques:

Journal: Life Science Alliance

Article Title: Inhibition of xanthine oxidoreductase with febuxostat, but not allopurinol, prevents inflammasome assembly and IL-1β release

doi: 10.26508/lsa.202403191

Figure Lengend Snippet: (A, B, C, D, E, F, G, H, I, J, K, L) Mouse bone marrow–derived macrophages were pre-treated for 30 min with DMSO vehicle (Veh), Fbx (200 µM), Allo (250 μg/ml), or Casp-i (Z-YVAD-FMK, 20 μM), followed by polarization with LPS (100 ng/ml) and IFNγ (20 ng/ml) for 18 h (A, B, C, D, E, F, G, H, I) or 2 h (J, K, L). (A) Frequency of CD80 and CD86 double-positive macrophages determined by flow cytometry. (B) Frequency of iNOS + macrophages by flow cytometry. (C) Schematic diagram representing breaks in TCA cycle promoted by LPS+γ polarization of macrophages. (D, E) Peak area comparison of itaconate and succinate obtained by liquid chromatography–mass spectrometry (LC-MS). (F) Schematic representation of the purine pathway. (G, H) Peak area comparison of hypoxanthine and xanthine obtained by liquid chromatography–mass spectrometry (LC-MS). (I) Uric acid levels were measured with a colorimetric assay according to the manufacturer’s instructions. (J, K) Cellular ROS and mitochondrial superoxide were determined using the probes CellROX and MitoSOX, respectively. Fluorescence was measured by flow cytometry. (L) NAD+ and NADH levels were measured using a luminescence assay according to the manufacturer’s instructions. Data are represented as the NAD/NADH ratio. Results are the mean ± SEM of at least three biological replicates. P -values were calculated (one-way ANOVA). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Red blood cells were lysed with red blood cell lysis buffer (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.3–7.4) for 3 min, and residual cells were plated in RPMI containing 10% FBS, 1% Pen/Strep, 1% HEPES (#SH3023701; Thermo Fisher Scientific), and 20% L929 media, for 7 d. After differentiation, macrophages were pre-treated with DMSO (# BP231-100; Thermo Fisher Scientific), FBX (200 μM; #S1547; Selleck Chemicals), Allo (250 μg/ml; #A8003; Sigma-Aldrich), or Casp-i (Z-YVAD-FMK, 20 μM; #218746; Sigma-Aldrich) for 30 min, and then with LPS (100 ng/ml; #L2630; Sigma-Aldrich) and IFNγ (20 ng/ml; #315-05; PeproTech), according to figure legends.

Techniques: Derivative Assay, Flow Cytometry, Comparison, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Colorimetric Assay, Fluorescence, Luminescence Assay

Journal: Life Science Alliance

Article Title: Inhibition of xanthine oxidoreductase with febuxostat, but not allopurinol, prevents inflammasome assembly and IL-1β release

doi: 10.26508/lsa.202403191

Figure Lengend Snippet: (B, C, D) Human monocyte-derived macrophages were pre-treated for 30 min with DMSO vehicle (Veh), Fbx (200 µM), Allo (250 μg/ml), or Casp-i (Z-YVAD-FMK, 20 μM), and then with LPS (100 ng/ml) and IFNγ (20 ng/ml) for 6 h (B, C) or 18 h (D). (B, C) ATP (1 mM) was added 3 h before harvesting. (A) Schematic representation of the differentiation of monocyte-derived macrophages. (B) IL-1β levels measured by ELISA. (C) Caspase-1 activity (relative luminescence units, RLU) was measured according to the manufacturer’s protocol. (D) Frequency of CD80 and CD86 double-positive macrophages determined by flow cytometry. Results are the mean ± SEM of at least four biological replicates. P -values were calculated using one-way ANOVA. * P < 0.05; ** P < 0.01.

Article Snippet: Red blood cells were lysed with red blood cell lysis buffer (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.3–7.4) for 3 min, and residual cells were plated in RPMI containing 10% FBS, 1% Pen/Strep, 1% HEPES (#SH3023701; Thermo Fisher Scientific), and 20% L929 media, for 7 d. After differentiation, macrophages were pre-treated with DMSO (# BP231-100; Thermo Fisher Scientific), FBX (200 μM; #S1547; Selleck Chemicals), Allo (250 μg/ml; #A8003; Sigma-Aldrich), or Casp-i (Z-YVAD-FMK, 20 μM; #218746; Sigma-Aldrich) for 30 min, and then with LPS (100 ng/ml; #L2630; Sigma-Aldrich) and IFNγ (20 ng/ml; #315-05; PeproTech), according to figure legends.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Flow Cytometry

Journal: Life Science Alliance

Article Title: Inhibition of xanthine oxidoreductase with febuxostat, but not allopurinol, prevents inflammasome assembly and IL-1β release

doi: 10.26508/lsa.202403191

Figure Lengend Snippet: Human monocyte-derived macrophages were pre-treated for 30 min with DMSO vehicle (Veh), Fbx (200 µM), Allo (250 μg/ml), or Casp-i (Z-YVAD-FMK, 20 μM), and then with LPS (100 ng/ml) and IFNγ (20 ng/ml) for 6 h. ATP (1 mM) was added 3 h before harvesting. (A) TNF-α levels measured by ELISA. (B) IL-6 levels measured by ELISA. P -values were calculated using one-way ANOVA. ** P < 0.01.

Article Snippet: Red blood cells were lysed with red blood cell lysis buffer (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.3–7.4) for 3 min, and residual cells were plated in RPMI containing 10% FBS, 1% Pen/Strep, 1% HEPES (#SH3023701; Thermo Fisher Scientific), and 20% L929 media, for 7 d. After differentiation, macrophages were pre-treated with DMSO (# BP231-100; Thermo Fisher Scientific), FBX (200 μM; #S1547; Selleck Chemicals), Allo (250 μg/ml; #A8003; Sigma-Aldrich), or Casp-i (Z-YVAD-FMK, 20 μM; #218746; Sigma-Aldrich) for 30 min, and then with LPS (100 ng/ml; #L2630; Sigma-Aldrich) and IFNγ (20 ng/ml; #315-05; PeproTech), according to figure legends.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: Life Science Alliance

Article Title: Inhibition of xanthine oxidoreductase with febuxostat, but not allopurinol, prevents inflammasome assembly and IL-1β release

doi: 10.26508/lsa.202403191

Figure Lengend Snippet: Mouse bone marrow–derived macrophages or human monocyte-derived macrophages were pre-treated for 30 min with DMSO vehicle (Veh), Fbx (200 µM), Allo (250 μg/ml), or Casp-i (Z-YVAD-FMK, 20 μM), followed by polarization with LPS (100 ng/ml) and IFNγ (20 ng/ml) for 6 h. (A, B, C, D) ATP (1 mM) was added 3 h before harvesting (A, B, C, D). (A) Fluorescence microscopy of ASC1 (red), DAPI (blue) in bone marrow–derived macrophages. ASC specks are indicated by white arrows. (B) Fluorescence microscopy of ASC1 (red), DAPI (blue) in human monocyte-derived macrophages. (C) Average number of detected specks per cell in mouse samples. (D) Average number of detected specks per cell in human samples. (C, D) Results are represented as total number of specks divided by the total number of cells per sample. No more than one speck per cell was observed. Results are representative of at least three biological replicates.

Article Snippet: Red blood cells were lysed with red blood cell lysis buffer (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.3–7.4) for 3 min, and residual cells were plated in RPMI containing 10% FBS, 1% Pen/Strep, 1% HEPES (#SH3023701; Thermo Fisher Scientific), and 20% L929 media, for 7 d. After differentiation, macrophages were pre-treated with DMSO (# BP231-100; Thermo Fisher Scientific), FBX (200 μM; #S1547; Selleck Chemicals), Allo (250 μg/ml; #A8003; Sigma-Aldrich), or Casp-i (Z-YVAD-FMK, 20 μM; #218746; Sigma-Aldrich) for 30 min, and then with LPS (100 ng/ml; #L2630; Sigma-Aldrich) and IFNγ (20 ng/ml; #315-05; PeproTech), according to figure legends.

Techniques: Derivative Assay, Fluorescence, Microscopy

Journal: Life Science Alliance

Article Title: Inhibition of xanthine oxidoreductase with febuxostat, but not allopurinol, prevents inflammasome assembly and IL-1β release

doi: 10.26508/lsa.202403191

Figure Lengend Snippet: Mouse bone marrow–derived macrophages or human monocyte-derived macrophages were pre-treated for 30 min with DMSO vehicle (Veh), Fbx (200 µM), Allo (250 μg/ml), or Casp-i (Z-YVAD-FMK, 20 μM), followed by polarization with LPS (100 ng/ml) and IFNγ (20 ng/ml) for 6 h. ATP (1 mM) was added 3 h before harvesting. (A) Fluorescence microscopy of ASC1 (red), NLRP3 (green), and DAPI (blue) in bone marrow–derived macrophages. ASC specks are indicated by white arrows. (B) Fluorescence microscopy of ASC1 (red), NLRP3 (green), and DAPI (blue) in human monocyte-derived macrophages. Results are representative of at least three biological replicates.

Article Snippet: Red blood cells were lysed with red blood cell lysis buffer (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.3–7.4) for 3 min, and residual cells were plated in RPMI containing 10% FBS, 1% Pen/Strep, 1% HEPES (#SH3023701; Thermo Fisher Scientific), and 20% L929 media, for 7 d. After differentiation, macrophages were pre-treated with DMSO (# BP231-100; Thermo Fisher Scientific), FBX (200 μM; #S1547; Selleck Chemicals), Allo (250 μg/ml; #A8003; Sigma-Aldrich), or Casp-i (Z-YVAD-FMK, 20 μM; #218746; Sigma-Aldrich) for 30 min, and then with LPS (100 ng/ml; #L2630; Sigma-Aldrich) and IFNγ (20 ng/ml; #315-05; PeproTech), according to figure legends.

Techniques: Derivative Assay, Fluorescence, Microscopy