Journal: PLoS ONE

Article Title: Omega-3 Free Fatty Acids Suppress Macrophage Inflammasome Activation by Inhibiting NF-κB Activation and Enhancing Autophagy

doi: 10.1371/journal.pone.0097957

Figure Lengend Snippet: LPS primed. (A) THP-1 cells and (B) BMDMs were stimulated with ATP. DHA (50 uM or 100 uM) added or not at the priming step. Supernatants were collected and an ELISA was performed to check IL-1β levels. (C) Western blot analysis of cell supernatants and lysates from LPS primed THP-1 cells treated with ATP in the presence or absence of DHA. The indicated samples were analyzed for mature IL-1β, pro- and mature caspase-1, ASC, NLRP3, and actin. (D) BMDMs were LPS primed and stimulated with the inflammasome inducers nigericin, Poly(dA:dT), and flagellin. DHA was added, or not, at the priming step. Supernatants were collected and IL-1β levels checked by ELISA. Results are representatives of a minimum of three independent experiments. * P<0.05, **P<0.001, ***P<0.0002, ****P<0.0001.

Article Snippet: The siRNA pools targeting human FFAR4 (sc-60737), GPR84 (sc-60751), or a control (sc-37007, Santa Cruz Biotechnology) were prepared with 2 µl HD Transfection Reagent (Promega) and used at a concentration of 40 nM to treat differentiated THP-1 cells overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: PLoS ONE

Article Title: Omega-3 Free Fatty Acids Suppress Macrophage Inflammasome Activation by Inhibiting NF-κB Activation and Enhancing Autophagy

doi: 10.1371/journal.pone.0097957

Figure Lengend Snippet: FFAR4-GFP expressing LPS primed non-differentiated THP-1 cells were stimulated with ATP or nigericin. DHA (100 µM) was added or not at the priming step. PFA fixed cells were permeablized and then stained with p65 antibody and analysis was performed using an ImageStream instrument. Shown are the (A) Imaging results and (B) Flow cytometry results. (C) ImageStream analysis of nuclear p65 NF-κB in non-differentiated THP-1 cells expressing FFAR1-GFP or FFAR4-GFP and exposed to LPS, or not, in the presence of DHA (50 µM), or not. (D) Confocal microscopy of BMDMs LPS primed and treated with nigericin in the presence, or absence of DHA, to examine the status of NF-κB translocation by p65 immunostaining. Shown are representative individual images. Scale bar is 20 µM. Whisker plot shows the amount of nuclear p65 immunofluorescence in the nuclei of BMDMs treated as indicated. Data are representative of three independent experiments. **P<0.001, n.s-not significant.

Article Snippet: The siRNA pools targeting human FFAR4 (sc-60737), GPR84 (sc-60751), or a control (sc-37007, Santa Cruz Biotechnology) were prepared with 2 µl HD Transfection Reagent (Promega) and used at a concentration of 40 nM to treat differentiated THP-1 cells overnight.

Techniques: Expressing, Staining, Imaging, Flow Cytometry, Confocal Microscopy, Translocation Assay, Immunostaining, Whisker Assay, Immunofluorescence

Journal: PLoS ONE

Article Title: Omega-3 Free Fatty Acids Suppress Macrophage Inflammasome Activation by Inhibiting NF-κB Activation and Enhancing Autophagy

doi: 10.1371/journal.pone.0097957

Figure Lengend Snippet: (A) Quantitative RT-PCR to detect Ffar1 , Gpr84 , and Ffar4 mRNA expression in BMDMs. Cells were LPS primed and ATP treated. DHA (100 µM) added during priming step, or not. Data is relative to Gapdh expression x 10 5 . Experiment performed 2 times with similar results. (B) Quantitative RT-PCR to detect GPR84 or FFAR4 mRNA in LPS primed THP-1 cells expressing siRNAs targeting FFAR4 or GPR84 , respectively. The results were normalized to β-actin expression and shown as relative to control. (C) IL-1β ELISA to assess inflammasome activity in FFAR1, GPR84, or FFAR4 knock-down cells. LPS primed THP-1 cells expressing siRNAs targeting FFAR1 , GPR84 , or FFAR4 were stimulated with ATP. DHA added during the priming step. Similar results from 3 experiments. *P<0.05, **P<0.001, ****P<0.0001.

Article Snippet: The siRNA pools targeting human FFAR4 (sc-60737), GPR84 (sc-60751), or a control (sc-37007, Santa Cruz Biotechnology) were prepared with 2 µl HD Transfection Reagent (Promega) and used at a concentration of 40 nM to treat differentiated THP-1 cells overnight.

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay