*2.6. Preparation of rHDL*

The rHDL particles were prepared using a previously described sodium cholate dialysis method [12,23]. In brief, the required amounts of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (Merck KGaA, Darmstadt, Germany), 1,2-dioleoyl-sn-glycero-3 phosphocholine (DOPC) (Merck KGaA, Darmstadt, Germany), or 1,2-dielaidoyl-sn-glycero-3-phosphocholine (elaidic acid PC) (Merck KGaA, Darmstadt, Germany), and cholesterol (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) were mixed and dried under an N2 gas stream. The dried mixture was dissolved in tris(hydroxymethyl)aminomethane (Tris)-buffered saline (TBS; 8.2 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 8.0) and supplemented with 19 mmol/L sodium deoxycholate until the solution was clear. ApoA1 from human plasma (Merck KGaA, Darmstadt, Germany) was added to the solution to make a final phosphatidylcholine (PC)–cholesterol–apoA1 molar ratio of 30:2:1. The mixture was incubated at 37 ◦C for 1 h and dialyzed against TBS for three days to remove sodium deoxycholate. The protein concentration was measured using the Bradford protein assay. The samples were subjected to non-denaturing 4–20% gradient polyacrylamide gel (Bio-Rad, Hercules, CA, USA) electrophoresis and stained with Coomassie Brilliant Blue to visualize the rHDL particles. Particle size was assigned by comparison with protein standards using a high molecular weight calibration kit (GE Healthcare, Madison, WI, USA).

#### *2.7. CUC Assay for rHDL*

rHDL was diluted in buffer to obtain a final apoA1 concentration of 1 μg/mL, and the CUC assay was performed with the HI-1000TM system as described above. To evaluate the effects of lecithin-cholesterol acyltransferase (LCAT) on the CUC assay, recombinant human LCAT (rhLCAT) (Sino Biological Inc., Beijing, China) or rhLCAT preincubated with 2 mM N-ethylmaleimide (NEM) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) at 30 ◦C for 30 min were mixed with rHDL to make a final rhLCAT–apoA1 molar ratio of 1.5:1 or 4.2:1, respectively, and incubated at 37 ◦C for 5 min. The samples were then diluted in buffer to obtain a final apoA1 concentration of 1 μg/mL, and the CUC assay was performed. The quantification of apoA1 was conducted using the HI-1000TM system and standardized using pooled serum. Briefly, an alkaline phosphatase-conjugated antiapoA1 mouse monoclonal antibody clone P1A5 (the preparation method is described in Appendix A) was added to rHDL captured by an anti-apoA1 mouse monoclonal antibody (8E10)-coated on magnetic particles and incubated at 37 ◦C for 10 min. After washing the particles with wash buffer, the chemiluminescent substrate was added and incubated at 42 ◦C for 5 min, and chemiluminescence was measured as a count. To improve inter- and intra-assay precision, the CUC per apoA1 value was used for CUC analysis of rHDL.

#### *2.8. Fluorescence-Based Assay for LCAT Activity*

A fluorescence-based assay for LCAT activity was developed according to a previous study [24]. The rHDL particles containing POPC or elaidic acid-PC, BODIPY-cholesterol (Avanti Polar Lipids, Alabaster, AL, USA), and apoA1 in a ratio of 30:2:1 were prepared and used as proteoliposome substrates. The samples were subjected to non-denaturing 4–20% gradient polyacrylamide gel (Bio-Rad, Hercules, CA, USA) electrophoresis and analyzed with a ChemiDoc Touch MP (Bio-Rad, Hercules, CA, USA) set at 488 nm for excitation and 520 nm for emission to detect BODIPY-cholesterol. The same gel was stained with Coomassie Brilliant Blue to visualize the rHDL particles.

The rhLCAT or rhLCAT preincubated with 2 mM NEM at 30 ◦C for 30 min was mixed with the proteoliposome substrates to make a final rhLCAT:apoA1 molar ratio of 0.5:1, and incubated at 37 ◦C for 10–90 min. The lipids were extracted from the samples, dissolved in 30 μL of chloroform, and applied to a thin-layer chromatography (TLC) silica gel 60 plate (Merck KGaA, Darmstadt, Germany), which was then placed into a closed glass tank and saturated with a developing solvent (petroleum ether, diethyl ether, and acetic acid in mole portions of 230:60:3). After 25 min, the TLC plate was removed from the tank and cholesterol spots and esterified cholesterol spots were detected using a ChemiDoc Touch MP set at 488 nm for excitation and 520 nm for emission. For quantitative analysis of cholesterol esterification rate, the TLC plate was exposed for 0.2 s and the fluorescence intensities of both cholesterol spots and esterified cholesterol spots were quantified by densitometry analysis using ImageJ® software (NIH, Bethesda, MD, USA). The cholesterol esterification rate was calculated using the following formula:

% Cholesterol esterification rate = (Fluorescence intensities of esterified BODIPY-cholesterol spots derived from each rHDL/Fluorescence intensities of BODIPY-cholesterol spots derived from rHDL without addition of rhLCAT) × 100.

> For visual inspection, exposure time for detecting BODIPY-cholesterol and esterified BODIPY-cholesterol spots were set to 0.2 and 3.0 s, respectively.
