Dugardin, Briand et al: Retrograde cholesterol transport in the human Caco-2/TC7 cell line: a model to study trans-intestinal cholesterol excretion in atherogenic and diabetic dyslipidemia

Supplemental Materials and Methods

Control of epithelial barrier integrity

In order to check the integrity of tight junctions at the end of the 6 hour-incubation period with plasma and phospholipid/bile salt micelles, Caco-2/TC7 cells were equilibrated for 10 minutes in HBSS – HEPES (Gibco) and Lucifer yellow (100µM – Sigma-Aldrich) was added on the apical side of the cells for 1 hour at 37°C. Apical and basolateral media were recovered and analysed by fluorescence spectrophotometry (λexcitation 432nm / λemission 538nm – Infinite 200 Pro NanoQuant, TECAN). The apparent permeability coefficient (Papp <1x10-6cm.sec-1) was determined according to the equation: Papp = J/A.C0, where J is the rate of appearance of the lucifer yellow in the basolateral compartment, C0 is the initial concentration in the apical medium and A, the surface area of the insert (4.2cm2). Papp was calculated to be 3.1 ± 2.9x10-7cm.sec-1, assessing the tight feature of the cellular monolayer and absence of paracellular leakage (28).

Cholesterol acceptor mixture preparation

Phospholipid/bile salt micelles containing 2mM phosphatidylcholine (PC)/10mM taurocholate (TC) were prepared as described (6). Briefly, for a final volume of 10mL, 200µL of a PC (100mM) stock solution in chloroform/methanol (2vol/1vol) were transferred in a glass bottle and dried using nitrogen gas. 4.2mL of a TC (24mM) stock solution, extemporaneously prepared in DMEM, and 5.8mL DMEM were added in the glass bottle, then sonicated (Ultrasonic cleaner) for 5 minutes and shaken at 37°C for 2 hours.

Incorporation of a cholesterol tracer in human plasma

Human plasma (total cholesterol: 230 ± 42mg/dL (Biomerieux enzymatic test)) was obtained from non-fasting healthy donors (French Blood Service), heat-inactivated, 0.22µm-filtered then distributed in aliquots and stored at -20°C until utilization. Labelling procedure was as follow: [3H]-cholesterol (42µCi per mg of plasma cholesterol) or nitrobenzoxadiazol-cholesterol (NBD-cholesterol) (1.6mg per mg of plasma cholesterol) was transferred in a glass tube and dried using nitrogen gas. Human plasma was added and incubated overnight at 37°C. The specific activity of tritiated plasma was ~14.9x106cpm per mg of cholesterol.

HDL preparation

Human plasma was obtained from non-fasting healthy donors (French Blood Service). HDL (d=1.12-1.21 g/ml) were prepared by sequential ultracentrifugation. Protein content was determined using Pierce™ BCA Protein Assay Kit (ThermoFisher).

Fluorescent microscopy analysis

Differentiated Caco-2/TC7 cells were incubated at 37°C for 6 hours with 2.5mL DMEM containing NBD-cholesterol-labelled plasma (1/40; vol/vol) in the basolateral compartment and 1.5mL of the indicated phospholipid/bile salt micelles in the apical compartment. Cells were then washed twice with ice-cold PBS, incubated with paraformaldehyde (PFA) 4% for 20 minutes then with L-lysine 0.1M for 1 hour. Fixed cells were permeabilized with 0.1% TritonX-100 in PBS at 37°C for 10 minutes. The transwell membrane was then detached from the insert, non-specific site blocked with Image-iT FX Signal Enhancer (Invitrogen), incubated overnight with primary anti-EEA1 antibody (BD Transduction Laboratories, ref 610456) then with secondary anti-mouse antibody (ThermoFisher, ref A10037) and mounted in fluorescent mounting medium (Dako) containing Hoechst 1:1,000 (Sigma-Aldrich). Preparations were analyzed with a Zeiss LSM 710 confocal microscope. Staining and colocalization Mander’s coefficients were quantified using Image J software (http://imagej.nih.gov/ij/, 1997-2016).

Post-nuclear cellular extract preparation and western blot analysis

Caco-2/TC7 cells were washed twice in PBS and harvested in lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail Complete (Roche) in PBS). Samples were sonicated for 5 minutes (Bioruptor, Diagenode). Homogenates were centrifuged (16,000g - 10min) and the supernatants stored at -20°C. Protein concentration was determined using the PierceTM BCA Protein Assay Kit (Thermo Scientific). 40µg of cellular proteins were separated on a 10% SDS-PAGE then transferred to a nitrocellulose membrane. The membrane was then incubated overnight with LDLR (LP02, Oncogene - 1:100) or β-Actin (SC-1616, Santa Cruz - 1:1,000) antibodies. After a 1 hour-incubation period with horseradish peroxydase-conjugated secondary antibodies (Sigma-Aldrich - 1:2,000), protein revelation was performed using the SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific) by chemiluminescence (Camera Gbox, SynGene) and band intensity was measured (GeneTools software, SynGene).