Journal: The FASEB Journal

Article Title: Monosodium urate crystals alter the circadian clock in macrophages leading to loss of NLRP3 inflammasome repression: Implications for timing of the gout flare

doi: 10.1096/fj.202202035r

Figure Lengend Snippet: FIGURE 4 The effect of monosodium urate (MSU) crystals on Caspase 1 activity and IL-1β secretion is time-of-day dependent. (A) Schematic of the experimental design to assess the time-of-day differences in NLRP3 inflammasome activity. Following 18 h serum starvation, cells were media changed to serum-replete media and either treated immediately with MSU crystals (500 μg/mL) or rested for 12 h before media-changing again with/without MSU crystal addition. Caspase 1 activity was measured following 12 h of MSU crystal exposure and normalized for differences in cell number as determined by CyQuant assay with reference to a standard curve. (B) The magnitude of Caspase 1 activity induced by MSU crystal exposure in cells at the 0–12 h compared with 12–24 h timepoints. (C) The magnitude of increase in secreted IL-1β levels induced by MSU crystal exposure in THP-1 macrophages at the 0–12 h compared with 12–24 h timepoints. Data shown are mean ± SEM. Data were analyzed by t-test with p < .05 considered statistically significant.

Article Snippet: Secreted IL- 1β protein levels were measured in the cell culture supernatant using a Duoset Human IL- 1β ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's protocol.

Techniques: Activity Assay, CyQUANT Assay

Journal: The FASEB Journal

Article Title: Monosodium urate crystals alter the circadian clock in macrophages leading to loss of NLRP3 inflammasome repression: Implications for timing of the gout flare

doi: 10.1096/fj.202202035r

Figure Lengend Snippet: FIGURE 6 BMAL1 represses expression of pro-IL-1β and CASP1 but not NLRP3 in THP-1 macrophages BMAL1 was knocked down or over-expressed in THP-1 macrophages using adenoviral-mediated gene delivery. Following gene transduction, cells were serum starved for 18 h then media changed to serum-replete media and either treated immediately with MSU crystals (500 μg/mL) (0–12 h timepoint) or rested for 12 h before media-changing again with/without MSU crystal addition (12–24 h timepoint). NLRP3 expression measured by RT-qPCR following BMAL1 knockdown at (A) the 0–12 h timepoint and (B) 12–24 h timepoint. NLRP3 expression measured by RT-qPCR following BMAL1 overexpression at (C) the 0–12 h timepoint and (D) 12–24 h timepoint. Pro-IL-1β expression measured by RT-qPCR following BMAL1 knockdown at (E) the 0–12 h timepoint and (F) 12–24 h timepoint. Pro-IL-1β expression measured by RT-qPCR following BMAL1 overexpression at (G) the 0–12 h timepoint and (H) 12–24 h timepoint. CASP1 expression measured by RT-qPCR following BMAL1 knockdown at (I) the 0–12 h timepoint and (J) 12–24 h timepoint. CASP1 expression measured by RT-qPCR following BMAL1 overexpression at (K) the 0–12 h timepoint and (L) 12–24 h timepoint. Data shown are mean ± SEM for 3 experimental replicates. Data were analyzed by one-way ANOVA (post-hoc Tukey) with p < .05 considered statistically significant.

Article Snippet: Secreted IL- 1β protein levels were measured in the cell culture supernatant using a Duoset Human IL- 1β ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's protocol.

Techniques: Expressing, Transduction, Quantitative RT-PCR, Knockdown, Over Expression

Journal: The FASEB Journal

Article Title: Monosodium urate crystals alter the circadian clock in macrophages leading to loss of NLRP3 inflammasome repression: Implications for timing of the gout flare

doi: 10.1096/fj.202202035r

Figure Lengend Snippet: FIGURE 7 REV-ERBα represses NLRP3 expression and inflammasome activity in THP-1 macrophages. Following 18 h serum starvation, cells were re-fed with serum-replete media and either treated immediately with/without MSU crystals (500 μg/mL) and heme (30 μM)), SR8278 (1 μM), heme+SR8278 or vehicle (0.1M NaOH + 0.1% DMSO) for 12 h (0–12 h timepoint) or rested for 12 h and then media-changed to fresh serum-replete media with/without MSU crystals and heme/SR8278/vehicle (12–24 h timepoint). (A) Caspase-1 activity normalized to cell number as measured at the 0–12 h timepoint and (B) 12–24 h timepoint. (C) Levels of secreted IL-1β measured by ELISA at the 0–12 h and (D) 12–24 h timepoint. To ensure all cells were exposed to the same vehicles, SR8278 vehicle (0.1%) DMSO) was added to the heme-only treatment and heme vehicle (0.1M NaOH) added to the SR8278-only treatment. Data shown are mean ± SEM for 3 experimental replicates. All data were analyzed by one-way ANOVA (post-hoc Tukey) p < .05 was considered statistically significant.

Article Snippet: Secreted IL- 1β protein levels were measured in the cell culture supernatant using a Duoset Human IL- 1β ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's protocol.

Techniques: Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: The negative cofactor 2 complex is a key regulator of drug resistance in Aspergillus fumigatus

doi: 10.1038/s41467-019-14191-1

Figure Lengend Snippet: a , b A549 epithelial cells or macrophages were infected with the wild-type (WT), the nctA null mutant ( ∆nctA ) or the nctA reconstituted isolate ( nctA rec ) for 24 h. Cytotoxicity of each mutant was evaluated by measuring the release of lactate dehydrogenase (LDH) activity into the culture medium. The data represent five different infection challenges with triplicate LDH activity measurements. Data are shown in fold change of LDH activity relative to the wild-type-infected cells. The error bars mean the standard error of the mean (SEM), and P- values were calculated by Kruskal–Wallis test with Dunn’s correction: * P < 0.0180; ** P < 0.0056 (for a , A549 cells). ** P < 0.0032; *** P < 0.0007 (for b , macrophages). c – f Granulocyte macrophage colony-stimulating factor-induced bone marrow-derived dendritic cells (gm-csf BMDCs) were infected with the A. fumigatus strains with the multiplicity of infection (MOI) = 5:1. Proinflammatory cytokines were quantified by ELISA. Data represent three biological replicates ( c – e ) or five biological replicates ( f ) with ± SEM. P- values were calculated by ANOVA with Tukey’s correction: *** P < 0.002; **** P < 0.0001. g , h Activation of dendritic cells measured by CD40 and CD80 markers by flow cytometry. Data represent three biological replicates with ±SEM. P -values were calculated by ANOVA: *** P < 0.0002; **** P < 0.0001 (for g ). *** P < 0.0004; **** P < 0.0001 (for h ). Source data are provided as a Source Data file.

Article Snippet: The concentration of IL-8 and IL-6 were determined in A549 epithelial cells co-cultured with A. fumigatus strains by using the Human IL-8/CXCL8 and IL-6 DuoSet ELISA according to the manufacturer’s instructions (R&D systems).

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