Figure 1│Nematic protein organisation technique (NPOT) for interactome analysis of evodiamine.
(a) The NPOT was performed under laminar flow and sterile condition at 4°C. 1 µM of evodiamine was mixed separately with 1 µg of total THP-1 extract and then subjected to NPOT heteroassemblies isolation. Experiments were performed three times independently. After the NPOT isolation step, the heteroassemblies were allowed to form overnight, and captured in 96 wells microtiter plates in the form of a droplet. Finally, the heteroassemblies are isolated and identified by mass spectroscopy directly in liquid.
(b) Capture of heteroassemblies in a droplet using 96 well microtiter plate. The floating heteroassemblies are circled with dotted line in three independent experiments. Non interacting proteins are precipitated to the bottom of the well in experiment 1 and 2. The latter is visible in the third experiment on the wall of the well as pointed with the red arrow. The latter is normally due to the vibration induced by incubator ventilation. In the absence of evodiamine no heteroassambly is formed (DMSO), as seen in the last picture on the right.

Figure 2 │Interactions between polyclonal anti-ABCA1 antibody, evodiamine and ABCA1 protein.
(a) Interaction of polyclonal anti-ABCA1 antibody with immobilized ABCA1 on CM5 sensor chip as assessed by Biacore 3000. The interaction affinity is calculated with association constant (KA) equal to 1.18e6M and dissociation constant (KD) equal to 4.57e-7M (antibody concentrations from bottom to top are ;4.25e-8, 8.5e-8,1.75e-7, 3.5e-7 and 7e-7M).
(b) Interaction affinity between evodiamine and ABCA1. Evodiamine interacts with an association constant (KA) equal to 1,23e6M and a dissociation constant (KD) equal to 8,1e-7M (evodiamine concentrations from bottom to top are; 2.5e-7, 5e-7 and 1e-6M). There is a bulk effect with Evodiamine at 1e-6M. This explains the higher signal (RU) in regard to the two lower concentrations.
(c) Interaction of retinal dehydogenase-1 (ALDHA1) with immobilized ABCA1. The two proteins interact with a KA equal to 1.25e6M and KD of 8.03e-7M (ALDHA1 concentrations from bottom to top are; 6e-8, 1.25e-7, 2.5e-7, 5e-7M and 1e-6M).

Figure 3 │ Effect of evodiamine (a) on ChE from THP-1 macrophages.
Differentiated THP-1 cells were loaded with [3H]-cholesterol together with the indicated treatments for 24 h. On the next day, the cells were washed twice with PBS and incubated with the same compounds [solvent vehicle control (Veh; ≤ 0.1% DMSO), evodiamine (1-20 μM), and the PPARγ agonist pioglitazone (10 μM) as positive control] with or without 10 µg/mL apo A1 (b) or 1% human plasma with 10 μM evodiamine (c) dissolved in serum-free medium for 6 h. Extracellular as well as intracellular radioactivity were quantified with scintillation counter. Differentiated THP-1-derived macrophages were treated with solvent vehicle control (Veh; ≤ 0.1% DMSO), evodiamine (10 μM), and the PPARγ agonist pioglitazone (10 μM) as positive control. After 24 h incubation, the cells were lysed and 20 μg protein was resolved via SDS-PAGE. Immunodetection was performed with antibodies against the indicated proteins, ABCA1 (d), ABCG1 (e), and SR-B1 (f), and visualized by chemiluminescence detection. All experiments were performed at least three times and data are presented as means ± S.D.  vs. solvent vehicle control, *p <0.05, **p <0.01, ***p <0.001, n.s. no significance (ANOVA / Bonferroni).

Figure 4 │Effect of evodiamine on ABCA1 transcription and the degradation rate of ABCA1 protein.
(a) Differentiated THP-1 macrophages were incubated with 10 μM evodiamine or 10 μM pioglitazone as positive control for 24 h. Total RNA was extracted and ABCA1 mRNA expression levels were quantified by qRT-PCR.
(b) 293T cells were transfected with a luciferase reporter construct driven by the human ABCA1 promoter as described in the Materials and Methods section. After transfection, cells were treated with 10 μM Evodiamine or 1 μM T0901317 as positive control for 24 h.
(c) Differentiated THP-1 macrophages were incubated for 24 h with (black circle) or without (Veh; white circle) evodiamine (10 μM) and lysed after addition of cycloheximide (CHX; 140 μM) at different time points (0, 10, 20, 40 min). Western blot analysis shows the decline of ABCA1 protein level with cycloheximide in the presence and absence of evodiamine. All data are means ± S.D. (n=3) vs. solvent vehicle control (DMSO), *p <0.05, **p <0.01, n.s. no significance (ANOVA / Bonferroni).

Figure 5 │Time dependent effect of evodiamine on ABCA1 protein expression.
(a) Differentiated THP-1 macrophages were incubated with solvent vehicle control (DMSO), 50 μg/mL digitonin, and indicated concentrations of evodiamine. After 24 h, the increased fluorescent signal was detected as a measure for cell viability.
(b) Differentiated THP-1 macrophages were incubated up to 24 h with (black circles) or without (Veh; white circles) evodiamine (10 μM). Cells were lysed at different time points (0, 3, 6, 15, 24 h) and 20 μg protein was resolved via SDS-PAGE. Immunodetection was performed with the antibody against ABCA1.  All data are means ± S.D. (n=3) vs. solvent vehicle control (DMSO) at certain time point, *p <0.05, ***p <0.001, n.s. no significance (ANOVA / Bonferroni).