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: Bioengineering

Article Title: Effect on Osteogenic Differentiation of Genetically Modified IL4 or PDGF-BB Over-Expressing and IL4-PDGF-BB Co-Over-Expressing Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro

doi: 10.3390/bioengineering8110165

Figure Lengend Snippet: Representative fluorescence microscopy images of mesenchymal stromal cells (MSCs) infected by IL-4 and/or PDGF-BB lentiviral vectors. GFP-MSCs, MSCs infected with control GFP-positive lentivirus vector; IL4-MSCs, MSCs infected with rIL-4 secreting GFP-positive lentivirus vector; PDGF-BB-MSCs, MSCs infected with hPDGF-BB secreting GFP-positive lentivirus vector; IL4-PDGF-BB-MSCs, previously established IL4-MSCs infected with hPDGF-BB secreting RFP-positive lentivirus vector.

Article Snippet: The expression levels of IL4 and PDGF-BB on day 3 were measured using the rabbit IL4 ELISA Kit (R&D Systems, Minneapolis, MN, USA) and the human PDGF-BB ELISA kit (R&D Systems, Minneapolis), respectively, according to the manufacturer’s protocols.

Techniques: Fluorescence, Microscopy, Infection, Control, Plasmid Preparation

Journal: Bioengineering

Article Title: Effect on Osteogenic Differentiation of Genetically Modified IL4 or PDGF-BB Over-Expressing and IL4-PDGF-BB Co-Over-Expressing Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro

doi: 10.3390/bioengineering8110165

Figure Lengend Snippet: The experimental outline of in vitro experiments. GFP-MSCs, IL4-MSCs, PDGF-BB-MSCs, or IL4-PDGF-BB-MSCs were seeded into T75 flasks and cultured in an MSC growth medium for 1 day. For the non-preconditioning MSC groups, the cells were cultured for 3 days with a fresh MSC growth medium. For the preconditioning MSC groups, cells were cultured in the MSC preconditioning medium for 3 days. The cells from all eight groups were washed three times with Dulbecco’s phosphate-buffered saline. The cells were trypsinized and seeded for each experiment, including cell proliferation, IL4 and PDGF-BB expression level, and osteogenic differentiation performed by alkaline phosphatase (ALP) staining and Alizarin red-staining. Abbreviations: MSCs, mesenchymal stromal cells; pMSCs, preconditioning MSCs; TNFα, tumor necrosis factor-alpha; LPS, lipopolysaccharide.

Article Snippet: The expression levels of IL4 and PDGF-BB on day 3 were measured using the rabbit IL4 ELISA Kit (R&D Systems, Minneapolis, MN, USA) and the human PDGF-BB ELISA kit (R&D Systems, Minneapolis), respectively, according to the manufacturer’s protocols.

Techniques: In Vitro, Cell Culture, Saline, Expressing, Staining

Journal: Bioengineering

Article Title: Effect on Osteogenic Differentiation of Genetically Modified IL4 or PDGF-BB Over-Expressing and IL4-PDGF-BB Co-Over-Expressing Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro

doi: 10.3390/bioengineering8110165

Figure Lengend Snippet: Fold change in the amount of dsDNA compared to day 1.

Article Snippet: The expression levels of IL4 and PDGF-BB on day 3 were measured using the rabbit IL4 ELISA Kit (R&D Systems, Minneapolis, MN, USA) and the human PDGF-BB ELISA kit (R&D Systems, Minneapolis), respectively, according to the manufacturer’s protocols.

Techniques:

Journal: Bioengineering

Article Title: Effect on Osteogenic Differentiation of Genetically Modified IL4 or PDGF-BB Over-Expressing and IL4-PDGF-BB Co-Over-Expressing Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro

doi: 10.3390/bioengineering8110165

Figure Lengend Snippet: ( A ) IL4 and PDGF-BB expression levels on day 3 in the IL4-MSC, IL4-pMSC, PDGF-BB-MSC, PDGF-BB-pMSC, IL4-PDGF-BB-MSC, and IL4-PDGF-BB-pMSC groups. IL4 expression levels on day 3 in the IL4-MSC, IL4-pMSC, IL4-PDGF-BB-MSC, and IL4-PDGF-BB-pMSC groups were 77.7 ± 4.4 ng/mL, 34.6 ± 5.7 ng/mL, 16.8 ± 9.4 ng/mL, and 41.8 ± 5.0 ng/mL, respectively. PDGF-BB expression levels on day 3 in the PDGF-BB-MSC, PDGF-BB-pMSC, IL4-PDGF-BB-MSC, and IL4-PDGF-BB-pMSC groups were 121.7 ± 20.1 pg/mL, 50.3 ± 20.4 pg/mL, 8.7 ± 2.0 pg/mL, 71.5 ± 7.8 pg/mL, respectively. ( B ) IL4 and PDGF-BB expression level-dsDNA ratios on day 3 in the IL4-MSC, IL4-pMSC, PDGF-BB-MSC, PDGF-BB-pMSC, IL4-PDGF-BB-MSC, and IL4-PDGF-BB-pMSC groups. IL4 expression level-dsDNA ratios on day 3 in the IL4-MSC, IL4-pMSC, IL4-PDGF-BB-MSC, and IL4-PDGF-BB-pMSC groups were 7.3 ± 0.6, 1.8 ± 0.2, 1.8 ± 1.0, and 2.3 ± 0.1, respectively. PDGF-BB expression level-dsDNA ratios on day 3 in the PDGF-BB-MSC, PDGF-BB-pMSC, IL4-PDGF-BB-MSC, and IL4-PDGF-BB-pMSC groups were 5.6 ± 0.9, 3.7 ± 1.4, 1.0 ± 0.3, and 3.9 ± 0.1, respectively.

Article Snippet: The expression levels of IL4 and PDGF-BB on day 3 were measured using the rabbit IL4 ELISA Kit (R&D Systems, Minneapolis, MN, USA) and the human PDGF-BB ELISA kit (R&D Systems, Minneapolis), respectively, according to the manufacturer’s protocols.

Techniques: Expressing

Journal: Bioengineering

Article Title: Effect on Osteogenic Differentiation of Genetically Modified IL4 or PDGF-BB Over-Expressing and IL4-PDGF-BB Co-Over-Expressing Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro

doi: 10.3390/bioengineering8110165

Figure Lengend Snippet: Alkaline phosphatase-staining in all eight groups 2 weeks after seeding. The percentages of stained area in the GFP-MSC, GFP-pMSC, IL4-MSC, IL4-pMSC, PDGF-BB-MSC, PDGF-BB-pMSC, IL4-PDGF-BB-MSC, and IL4-PDGF-BB-pMSC groups were 19.8 ± 5.3%, 28.3 ± 1.4%, 4.3 ± 0.7%, 6.0 ± 2.4%, 44.2 ± 11.7%, 42.5 ± 8.1%, 21.4 ± 1.5%, and 25.7 ± 1.0%, respectively. Abbreviations: ALP, alkaline phosphatase.

Article Snippet: The expression levels of IL4 and PDGF-BB on day 3 were measured using the rabbit IL4 ELISA Kit (R&D Systems, Minneapolis, MN, USA) and the human PDGF-BB ELISA kit (R&D Systems, Minneapolis), respectively, according to the manufacturer’s protocols.

Techniques: Staining

Journal: Bioengineering

Article Title: Effect on Osteogenic Differentiation of Genetically Modified IL4 or PDGF-BB Over-Expressing and IL4-PDGF-BB Co-Over-Expressing Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro

doi: 10.3390/bioengineering8110165

Figure Lengend Snippet: Alizarin red-staining in all eight groups 4 weeks after seeding. The percentages of stained area in the GFP-MSC, GFP-pMSC, IL4-MSC, IL4-pMSC, PDGF-BB-MSC, PDGF-BB-pMSC, IL4-PDGF-BB-MSC, and IL4-PDGF-BB-pMSC groups were 33.1 ± 4.7%, 45.1 ± 4.9%, 0.64 ± 0.08%, 0.58 ± 0.03%, 51.7 ± 14.5%, 40.7 ± 6.7%, 17.3 ± 2.5%, and 41.2 ± 1.7%, respectively.

Article Snippet: The expression levels of IL4 and PDGF-BB on day 3 were measured using the rabbit IL4 ELISA Kit (R&D Systems, Minneapolis, MN, USA) and the human PDGF-BB ELISA kit (R&D Systems, Minneapolis), respectively, according to the manufacturer’s protocols.

Techniques: Staining

Journal: Bioengineering

Article Title: Effect on Osteogenic Differentiation of Genetically Modified IL4 or PDGF-BB Over-Expressing and IL4-PDGF-BB Co-Over-Expressing Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro

doi: 10.3390/bioengineering8110165

Figure Lengend Snippet: The summary of the results.

Article Snippet: The expression levels of IL4 and PDGF-BB on day 3 were measured using the rabbit IL4 ELISA Kit (R&D Systems, Minneapolis, MN, USA) and the human PDGF-BB ELISA kit (R&D Systems, Minneapolis), respectively, according to the manufacturer’s protocols.

Techniques: Expressing