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: The effect of Oxy210 on reverse cholesterol transport. A: The effect of Oxy210 (5 μM) on the abundance of total and surface ABCA1 and ABCG1 in BMDM. Four biological replicates are shown in each Western blot. B: Quantitation (densitometry) of the abundance of total ABCA1 in BMDM as shown in (A) . C: Quantitation (densitometry) of the abundance of surface ABCA1 in BMDM as shown in A. D: Quantitation (densitometry) of the abundance of ABCG1 in BMDM as shown in (A). E: The effect of Oxy210 (5 μM) on the abundance of SRB1 in BMDM. Four biological replicates are shown in each western blot. F: Quantitation (densitometry) of the abundance of SRB1 in BMDM as shown in (E). G: The effect of Oxy210 (5 μM) on cholesterol efflux from BMDM 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. H: The effect of Oxy210 (5 μM) on cholesterol efflux from BMDM to HDL. Cells were labeled with [ 3 H] cholesterol, proportion of the label moved from cells to HDL (30 μg/ml) over subsequent 2 h incubation is shown. I: Dose-dependence of the effect of Oxy210 on cholesterol efflux from RAW264.7 cells 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. J: The effect of Oxy210 (5 μM) and LXR agonist TO-901317 (4 μM) on the abundance of ABCA1 in RAW264.7 cells. Quantitated by densitometry of western blots. K: The effect of Oxy210 (5 μM) and LXR agonist TO-901317 (4 μM) on cholesterol efflux to apoA-I (20 μg/ml) from RAW 264.7 cells. Quantitated as described in (G). L: The effect of Oxy210 (5 μM), LXR antagonist GSK2033 (1 μM), and LXR agonist TO-901317 (4 μM) on cholesterol efflux to apoA-I (20 μg/ml) from RAW 264.7 cells. Quantitated as described in (G) . M: The effect of Oxy210 (5 μM) and LXR agonist TO-901317 (4 μM) on the expression of LXR-dependent and LXR-independent genes in BMDM (RT -PCR). N: The effect of Oxy210 (5 μM) on lipid raft abundance in ABCA +/+ and ABCA1 −/− fibroblasts. 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 . O: The effect of Oxy210 (5 μM) on the rate of cholesterol biosynthesis in BMDM ([ 3 H] acetate incorporation in cholesterol over 4 h). All graphs: Mean ± SEM are shown. ∗ P < 0.05, ∗∗ P < 0.01; ∗∗∗ P < 0.001 versus vehicle ( t -test). ## P < 0.01 versus TO-901317 (ANOVA). BMDM, bone marrow-derived macrophage; LXR, liver X receptor; apoA-I, apolipoprotein A-I; ABCG1, ATP binding cassette transporter G1; ABCA1, ATP binding cassette transporter A1.

Article Snippet: Antibodies used in western blot analysis:ATP binding cassette transporter A1 (ABCA1) (Abcam, #ab18180, 1:3000), ATPase (Abcam, #ab76671, 1:5000), GAPDH (Merck, #CB1001, 1:5000), ARF GTPase 6 (Arf6) (Sigma-Aldrich, #A5230, 1:2000), cell division cycle control protein 42 (Cdc42) (BD transduction Laboratories, #610929, 1:500), mitogen-activated protein kinase 1/2 (Erk1/2) (Millipore, #66192, 1:10,000), pErk1/2 (Cell Signaling Technology, #4370, 1:2000), TMS1(Cell Signaling Technology, #67824 1:1000), NRLP3 (Cell Signaling Technology, #15101, 1:1000), Caspase-1 ((Cell Signaling Technology, #24232, 1:1000), ATP binding cassette transporter G1 (ABCG1) (Novus Biologicals, #NB400-132, 1:1000), Anti-mouse HRP-linked antibody (Cell Signaling Technology, #7076, 1:5000), Anti-rabbit HRP-linked antibody (Cell Signaling Technology, #7074, 1:5000).

Techniques: Western Blot, Quantitation Assay, Labeling, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction, Clinical Proteomics, Isolation, Gradient Centrifugation, MANN-WHITNEY, Derivative Assay, Binding Assay

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: Schematic representation of the proposed mechanism of action of Oxy210. Plasma membrane components are constantly recycling between the membrane and intracellular compartments. During recycling they pass through a checkpoint in late endosomes, where components of “disordered” regions of plasma membrane are directed to lysosomes, while “ordered” ensembles are directed to the plasma membrane lipid rafts. Small GTPases, including Arf6, are proposed to play key role in this regulatory checkpoint through activation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K), which catalyzes conversion of PIP1 to PIP2. Arf6 is also involved in sorting of internalized ABCA1; however, it works in an opposite direction trafficking ABCA1 for degradation in lysosomes. We propose that Oxy210 reduces the abundance and activity of Arf6, which in turn decreases the activity of PIP5K, the abundance of PIP2 and the recycling of lipid rafts to the plasma membrane. Suppression of Arf6 by Oxy210 also inhibited internalization and degradation of ABCA1, increasing its abundance on the cell surface and consequently increasing the rate of cholesterol efflux. ABCA1, ATP binding cassette transporter A1; Arf6, ARF GTPase 6.

Article Snippet: Antibodies used in western blot analysis:ATP binding cassette transporter A1 (ABCA1) (Abcam, #ab18180, 1:3000), ATPase (Abcam, #ab76671, 1:5000), GAPDH (Merck, #CB1001, 1:5000), ARF GTPase 6 (Arf6) (Sigma-Aldrich, #A5230, 1:2000), cell division cycle control protein 42 (Cdc42) (BD transduction Laboratories, #610929, 1:500), mitogen-activated protein kinase 1/2 (Erk1/2) (Millipore, #66192, 1:10,000), pErk1/2 (Cell Signaling Technology, #4370, 1:2000), TMS1(Cell Signaling Technology, #67824 1:1000), NRLP3 (Cell Signaling Technology, #15101, 1:1000), Caspase-1 ((Cell Signaling Technology, #24232, 1:1000), ATP binding cassette transporter G1 (ABCG1) (Novus Biologicals, #NB400-132, 1:1000), Anti-mouse HRP-linked antibody (Cell Signaling Technology, #7076, 1:5000), Anti-rabbit HRP-linked antibody (Cell Signaling Technology, #7074, 1:5000).

Techniques: Clinical Proteomics, Membrane, Activation Assay, Activity Assay, Binding Assay