Journal: Journal of Lipid Research
Article Title: Targeting the ARF6-dependent recycling pathway to alter lipid rafts and reduce inflammation
doi: 10.1016/j.jlr.2025.100900
Figure Lengend Snippet: Mechanism of the effects of Oxy210 in RAW264.7 cells. A: The effect of Arf6 silencing and of Oxy210 (5 μM) on lipid raft abundance. Plasma membranes isolated from cells labeled with [ 3 H] cholesterol were fractionated by density gradient centrifugation as described in “Methods”. Lipid rafts were defined as fractions with highest [ 3 H] cholesterol and flotillin-1 content; P < 0.05 (Mann-Whitney test), Area under the curve values are shown in . B: The effect of Arf6Q67L overexpression and of Oxy210 (5 μM) on lipid raft abundance. Methodology is the same as in A, P < 0.001 (Mann-Whitney test). Area under the curve values are shown in . C: The effect of Arf6 silencing and overexpression and Oxy210 (5 μM) on cholesterol efflux to apoA-I. Cells were labeled with [ 3 H] cholesterol, proportion of the label moved from cells to apoA-I (20 μg/ml) over subsequent 2 h incubation is shown. Mean ± SEM; ∗ P < 0.05; versus siRNA scr or pCMV; (ANOVA, n = 4). D: The effect of Arf6 overexpression and Oxy210 (5 μM) on the ABCA1 abundance. The ABCA1 abundance was quantified by densitometry of western blots (shown in B) and was normalized to the abundance of ABCA1 in untransfected cells. Mean ± SEM; ∗∗ P < 0.01; versus pCMV; (ANOVA, n = 3). E: The effect of Arf6 silencing and Oxy210 (5 μM) on LPS-induced secretion of IL6. After Arf6 silencing, cells were stimulated with LPS (100 ng/ml) for 20 h in the presence or absence of Oxy210; medium was collected and subjected to analysis. Arrows depict the level of inhibition by Oxy210. Mean ± SEM;∗∗ P < 0.01 versus vehicle, ∗∗∗ P < 0.001 versus vehicle, ### P < 0.001 versus siRNA scr ; (n = 4, t -test). F: The effect of Arf6 silencing and Oxy210 (5 μM) on LPS-induced secretion of TNFα. After Arf6 silencing, cells were stimulated with LPS (100 ng/ml) for 20 h in the presence or absence of Oxy210; medium was collected and subjected to analysis. Arrows depict the level of inhibition by Oxy210. Mean ± SEM;∗∗ P < 0.01 versus vehicle, ∗∗∗ P < 0.001 versus vehicle, ### P < 0.001 versus siRNA scr ; (n = 4, t -test). G: The effect of Oxy210 (5 μM) on the abundance of Arf6. Quantitated by densitometry of western blots shown in C. Mean ± SEM; ∗∗ P < 0.01; versus vehicle (n = 4, t -test). H: The effect of Oxy210 (5 μM) on the ratio of phosphorylated to total Arf6. Mean ± SEM; ∗∗ P < 0.01; versus vehicle (n = 4, t -test). Quantitated by densitometry of western blots shown in D. I: Binding of Arf6 to Oxy210 in protein lipid overlay assay. Indicated amounts of Oxy210, Arf6 (0.2 μg) or vehicle (TBS) were applied (2 μl/dot) on a nitrocellulose membrane (left) or a commercial membrane with preloaded lipids was used (right). Both membranes were incubated for 1 h in the blocking solution and then overnight at 4 ° C with recombinant human His-Arf6 (1 μg/ml), followed by 2 h incubation with anti-His Tag monoclonal antibody (1:1000). J: The effect of Oxy210 (5 μM) on the abundance of PIP1 and PIP2. Lipids extracted from RAW264.7 cell whole cell lysate or isolated lipid rafts were analyzed using liquid chromatography/mass-spectrometry (lipidomics) as described in “Methods”. All concentrations were normalized to the level of PC. Mean ± SEM; ∗ P < 0.05; versus vehicle (n = 4, t -test). LPS, lipopolysaccharide; TNFα, tumor necrosis factor α; apoA-I, apolipoprotein A-I; ABCA1, ATP binding cassette transporter A1; Arf6, ARF GTPase 6.
Article Snippet: Arf6 activation pull-down assay (Cytoskeleton, #BK033-S)) was implemented according to the manufacturer’s protocol.
Techniques: Clinical Proteomics, Isolation, Labeling, Gradient Centrifugation, MANN-WHITNEY, Over Expression, Incubation, Western Blot, Inhibition, Binding Assay, Protein-lipid Overlay Assay (PLOA), Membrane, Blocking Assay, Recombinant, Liquid Chromatography, Mass Spectrometry
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![Mechanism of the effects of Oxy210 in RAW264.7 cells. A: The effect of <t>Arf6</t> silencing and of Oxy210 (5 μM) on lipid raft abundance. Plasma membranes isolated from cells labeled with [ 3 H] cholesterol were fractionated by density gradient centrifugation as described in “Methods”. Lipid rafts were defined as fractions with highest [ 3 H] cholesterol and flotillin-1 content; P < 0.05 (Mann-Whitney test), Area under the curve values are shown in . B: The effect of Arf6Q67L overexpression and of Oxy210 (5 μM) on lipid raft abundance. Methodology is the same as in A, P < 0.001 (Mann-Whitney test). Area under the curve values are shown in . C: The effect of Arf6 silencing and overexpression and Oxy210 (5 μM) on cholesterol efflux to apoA-I. Cells were labeled with [ 3 H] cholesterol, proportion of the label moved from cells to apoA-I (20 μg/ml) over subsequent 2 h incubation is shown. Mean ± SEM; ∗ P < 0.05; versus siRNA scr or pCMV; (ANOVA, n = 4). D: The effect of Arf6 overexpression and Oxy210 (5 μM) on the ABCA1 abundance. The ABCA1 abundance was quantified by densitometry of western blots (shown in B) and was normalized to the abundance of ABCA1 in untransfected cells. Mean ± SEM; ∗∗ P < 0.01; versus pCMV; (ANOVA, n = 3). E: The effect of Arf6 silencing and Oxy210 (5 μM) on LPS-induced secretion of IL6. After Arf6 silencing, cells were stimulated with LPS (100 ng/ml) for 20 h in the presence or absence of Oxy210; medium was collected and subjected to analysis. Arrows depict the level of inhibition by Oxy210. Mean ± SEM;∗∗ P < 0.01 versus vehicle, ∗∗∗ P < 0.001 versus vehicle, ### P < 0.001 versus siRNA scr ; (n = 4, t -test). F: The effect of Arf6 silencing and Oxy210 (5 μM) on LPS-induced secretion of TNFα. After Arf6 silencing, cells were stimulated with LPS (100 ng/ml) for 20 h in the presence or absence of Oxy210; medium was collected and subjected to analysis. Arrows depict the level of inhibition by Oxy210. Mean ± SEM;∗∗ P < 0.01 versus vehicle, ∗∗∗ P < 0.001 versus vehicle, ### P < 0.001 versus siRNA scr ; (n = 4, t -test). G: The effect of Oxy210 (5 μM) on the abundance of Arf6. Quantitated by densitometry of western blots shown in C. Mean ± SEM; ∗∗ P < 0.01; versus vehicle (n = 4, t -test). H: The effect of Oxy210 (5 μM) on the ratio of phosphorylated to total Arf6. Mean ± SEM; ∗∗ P < 0.01; versus vehicle (n = 4, t -test). Quantitated by densitometry of western blots shown in D. I: Binding of Arf6 to Oxy210 in protein lipid overlay assay. Indicated amounts of Oxy210, Arf6 (0.2 μg) or vehicle (TBS) were applied (2 μl/dot) on a nitrocellulose membrane (left) or a commercial membrane with preloaded lipids was used (right). Both membranes were incubated for 1 h in the blocking solution and then overnight at 4 ° C with recombinant human His-Arf6 (1 μg/ml), followed by 2 h incubation with anti-His Tag monoclonal antibody (1:1000). J: The effect of Oxy210 (5 μM) on the abundance of PIP1 and PIP2. Lipids extracted from RAW264.7 cell whole cell lysate or isolated lipid rafts were analyzed using liquid chromatography/mass-spectrometry (lipidomics) as described in “Methods”. All concentrations were normalized to the level of PC. Mean ± SEM; ∗ P < 0.05; versus vehicle (n = 4, t -test). LPS, lipopolysaccharide; TNFα, tumor necrosis factor α; apoA-I, apolipoprotein A-I; ABCA1, ATP binding cassette transporter A1; Arf6, ARF GTPase 6.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2991/pmc12552991/pmc12552991__gr6.jpg)
