SUPPORTING INFORMATION
MATERIALS AND METHODS
Materials. ES cell culture media (DMEM, Penicillin/Streptomycin, non-essential amino acids and Lglutamine) were obtained from Invitrogen. Serum suitable for ES cell culture was purchased from
Multicell Technology (Lincoln, RI). Leukemia Inhibitor Factor (LIF) was home made. R1 ES cells were
a generous gift of Dr A. Nagy (Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto,
Ontario, Canada). Media for embryo culture and injection (M2 and M16) were provided by Eurogentec
(Liège, Belgium). Cangrelor, formerly denoted as AR-C69931MX, was provided by The Medicines
Company (New Jersey, USA) and was used as previously described (1)
Generation of P2Y13-deficient mice. A ~9kb DNA fragment containing the P2Y13 gene was cloned
from a 129/Sv λZAP genomic library (obtained from S. Refetoff, University of Chicago, Chicago, IL).
To generate the targeting construct, a Sal1-XbaI fragment of ~8.,5kb, with the XbaI recognition site
located ~1kb upstream the ATG starting codon of the mouse P2Y13 gene (NM_028808), was cloned in
the pFlox vector (Figure 1A, targeting construct). A HindIII-AccI (AccI was blunted for cloning purpose)
fragment of 3.3kb was inserted in the same vector at the other edge of the neomycin resistance
cassette.
Upon integration of the targeting construct by homologous recombination into ES cell genomic DNA, a
1.2kb fragment containing the first non-coding exon, the only intron and the first 182bp from the ATG
starting coding sequence was replaced by the neo-cassette.
After electroporation of the linearized targeting construct into R1 ES cells (2), G-418-resistant ES cell
colonies were isolated and analyzed by southern blotting. Genomic DNA was restricted with EcoRI
and screened using an internal (Neo) and an external probe (Figure 1A-B). Clones selected for
aggregation were those characterized in southern blotting by one 6.0kb and one 4.9kb fragment when
analyzed with the external probe and only a 4.9kb fragment with the internal one. One example of a
positive ES cell clone is indicated by a white arrow (Figure 1B). A total of 716 ES cell clones were
screened and 3.2 % of them turned out to be positive.
To generate chimeric mice, ES cells were aggregated overnight with CD1 eight-cell morulae. The next
day, the resulting blastocysts were implanted in pseudopregnant CD1 females. Fifty-four chimeras
(mostly males) were obtained from 9 different P2Y13-targeted ES cell clones. Male chimeras obtained
from two different ES cell colonies were used to generate the P2Y13-deficient mouse strain. P2Y13
mutant mice were further characterized by southern blotting (Figure 1C) and PCR (Figure 1D). Primers
a-b-c (sequences in supporting Table 1) were routinely used to genotype the mice.
To confirm the deletion of the P2Y13 gene, we assayed its expression, by RT-PCR, in the liver where
expression has been reported (3). The primers (1-2, see Figure 1A and supporting Table 1) were set
up so as to detect any residual P2Y13 mRNA. Each reaction was carried out with or without (data not
shown) a reverse transcription step to ensure that the amplified product was not due to contaminant
DNA. The expected 525bp signal was detected in tissues from control wild-type littermates but not in
P2Y13 (-/-) ones, indicating that the targeting construct completely abolished P2Y13 mRNA expression.
HPRT transcript, in contrast, was detected in all tissues independently of genotype (Figure 1E). It was
checked that the targeting of the P2Y13 gene did not interfere with the function of the adjacent P2Y12
gene (unpublished data).
The P2Y13 mutation was crossed into the C57BL/6 genetic background for 10 generations. All of the
genotypes were obtained at the expected ratios. P2Y13-deficient mice were indistinguishable from
control mouse littermates. They were fertile, they grew up similarly to wild-type mice and their behavior
was normal. Gross examination of internal organs did not reveal any abnormality.
Animals and Diets. SR-BIflox/fox/Cre mice (namely hypomSR-BIKOliver), which were deficient for SRBI in hepatocytes, were obtained in the homogeneous C57BL/6 genetic background as described (4).
The Alb-Cre transgene was systematically maintained in a hemizygous state throughout the study.
Mice heterozygotic for P2Y13 were intercrossed to obtain P2Y13 (+/+) and P2Y13 (-/-) littermates. The
animals were caged in animal rooms with alternating 12h periods of light (07:00-19:00) and dark
(19:00-07:00). There were weaned at 21 days and fed ad libitum a normal mouse chow diet (R04-10,
SAFE France). All animal procedures were in accordance with the guidelines of the Committee on
Animals of the Midi-Pyrénées Ethics Committee on Animal Experimentation and with the French
Ministry of Agriculture license.
RNA isolation and quality control. After mechanical grinding, total RNA was extracted from each
liver sample using the RNeasy® Mini Kit (Qiagen). Genomic DNA contamination was removed using
RNase-free DNase set (Qiagen). The RNA was then eluted in DNase RNase-free water and finally the
concentration of each sample was measured at a wavelength of 260 nm. The quality of the RNA was
checked by capillary electrophoresis (Agilent® RNA Nano chip).
Reverse transcription. Reverse transcription of mRNA was performed using Susperscript ® III
Reverse Transcriptase (Invitrogen) in the presence of primer p(dT) (Roche Diagnostics) and RNase
OUT™ (Invitrogen). Briefly, 5 µg of total RNA from each sample were used with 2 µL of primer p(dT) 15
(20 µmol/L) and 0.5 µL of RNase OUT. After 8 minutes of incubation at 70°C to inactivate DNase, 100
units of Superscript III Reverse Transcriptase (Invitrogen) were added and the mixture incubated at
25°C for 10 minutes and then at 50°C for 1 hour. The RT reaction was ended by heating the mixture at
95°C for 5 minutes, it was then chilled and stored at -80°C until use.
Analysis of Gene Expression by real-time quantitative PCR. Real-time quantitative PCR analysis
was performed using SYBR Green I Master (Roche) on LightCycler ® 480 Real-Time PCR System.
Liver expression of each gene was studied and compared to the expression of the housekeeping
genes h. ypoxanthine phosphorybosyltransferase (HPRT) and TATA-binding protein (TBP). PCR
reactions were run in a 96-well format with 20 µL reaction mixture containing SYBR Green I Master
(10 µL), cDNA from the RT reaction (5 g, 4 µL) and gene specific primers (900 nM, 6 µL). Reactions
were run with a standard 2-step cycling program, 95°C for 10 seconds and 60°C for 40 seconds, for
40 cycles. Each analysis was performed in triplicate and included both housekeeping genes and the
gene of interest, primer/probe pairs as multiplexed samples. Comparison of gene expression values
between wild-type and P2Y13KO mice was done with the use of the Student ‘s T-test (2 tailed). Data
are expressed as fold change  SEM against P2Y13 (+/+) mice and are normalized to HPRT (n8 mice
per group). The threshold for fold-change was set above 1.5 or bellow 0.5 and **, p<0.01. Results are
representative of two independent experiments. The specific primers used are reported in supporting
Table 2.
Perfused mouse liver experiments. Mice 10-15 weeks old were anesthetized by intraperitoneal
injection of ketamine hydrochloride and xylazine hydrochloride. The liver was perfused in situ for 10
min at 37°C with PBS medium (Invitrogen) containing 50 µg proteins/mL of
125I-HDL
3
(specific
radioactivities ranged form 1000-1500 cpm/ng of proteins) with or without cangrelor (10 µM). Livers
were extensively washed at 4°C in PBS and radioactivity associated with the liver was counted (n  6
per group).
Isolation of primary mouse hepatocytes and confocal microscopy visualization of fluorescent
HDL endocytosis. 10-15 weeks old mice were anesthetized as described above, liver were perfused
and hepatocytes isolated as previously described (5) then seeded on collagen-coated coverslip. To
visualize fluorescent HDL endocytosis, 50 g/mL DyLight549-HDL particles were incubated with
primary hepatocytes for 30 min at 37°C. Following washes, cells were fixed (4% paraformaldehyde, 20
min, 4°C) then examined using a Zeiss LSM 510 META confocal microscope equipped with a 63x
Plan-Apochromat objective.
Plasma lipid analyses. Blood samples were collected from 3h-fasted mice. Total cholesterol, total
triglycerides, and HDL levels were measured with commercial kits (CHOD-PAP for cholesterol and
GPO-PAP for triglycerides; BIOLABO SA, Maizy, France). HDLs were isolated by LDL/VLDL
precipitation with phosphotungstate/MgCl2 reagent (HDL-cholesterol, BIOLABO SA, Maizy, France).
Hepatic lipid analyses. Following homogenization of liver samples in methanol/5mM EGTA (2:1 v/v),
lipids corresponding to an equivalent of 1 mg tissue were extracted in chloroform/methanol/water
(2.5:2.5:2.1, v/v/v), in the presence of the internal standard: 3 g of stigmasterol, 3 g of cholesteryl
heptadecanoate and 6 g of glyceryl triheptadecanoate (6). Neutral lipids were analyzed by gas-liquid
chromatography on a FOCUS Thermo Electron system using a Zebron-1 Phenomenex fused silica
capillary columns (5 m x 0.32 mm i.d., 0.50 mm film thickness). Oven temperature was programmed to
ramp from 200°C to 350°C at 5°C per min and the carrier gas was hydrogen (0.5 bar). The injector
was held at 315°C and the detector at 345°C (7).
Cannulation of the Common Bile Duct and Collection of Hepatic Bile. Male mice 10-15 weeks old
were fasted for 3 hours and than were anesthetized by intra-peritoneal injection of ketamine
hydrochloride and xylazine hydrochloride. The common bile duct was ligated close to the duodenum;
and then the gallbladder was punctured and cannulated with a polyethylene-10 catheter. After 30 min,
bile collection was performed for 210 min in cangrelor injection studies or for 60 min otherwise, and
bile flow (µL/min/100 g of body weight) was determined gravimetrically assuming a density of 1 g/mL
for bile.
Biliary lipid analyses. Bile acids were determined by HPLC. Briefly, 8 µL of bile were diluted with 10
µL of internal standard (norcholic acid 9.2 mM; Steraloids, Wilton, NH) in methanol to precipitate
proteins. Samples were centrifuged at 14000 rpm for 10 min at 4°C, and the supernatant was further
diluted in methanol (1:60). Samples (20 µL) were separated by HPLC (DIONEX Sumit) on a
LiChrosorb RP-18 analytical column (5 µm particle size, 250x4.6 mm). This was fitted with a C18
guard column cartridge (10x4.6 mm; Supelco) and coupled to a light scattering detector (Polymer
Laboratory ELS 2100, nitrogen flow 1.2 mL/min, evaporating temperature 90°C, and nebulizer
temperature 70°C). Separation was achieved at a flow rate of 0.7 mL/min using a ternary solvent
system (8) as follows: ammonium acetate, pH 5.2 (30 mM), methanol, acetonitrile, 35:60:5 v/v/v for 25
min move to 24:53:23 in 5 min, then plateau for 20 min. Phospholipids were determined by HPLC.
Briefly, 5 µL of bile were extracted as previously described (6). Lipid extracts were analyzed by HPLC
(DIONEX Sumit) on a Uptisphere6OH analytical column (5 µm particle size, 250 x 2.1 mm) fitted with a
DIOL guard column cartridge (10 x 2.1mm mm; INTERCHIM) and coupled to a light scattering detector
(Polymer Laboratory ELS 2100, nitrogen flow 1.8 mL/min, evaporating temperature 50°C, and
nebulizer temperature 80°C). Separation was achieved at a flow rate of 0.25 mL/min using a gradient
of B (isopropanol:water:triethylamin:acetic acid (85/15/0.014/0.5 v/v/v/v) in A (Hexane:isopropanol :
Triethylamin:acetic acid (82/18/0.014/0.5 v/v/v/v) from 5 to 35% of B in 35 min.
Cholesterol was determined by GC. Briefly, 5 µL of bile were extracted in the presence of stigmasterol
(2 µg) then quantified as previously described (6).
In vivo macrophage-to-feces RCT. The in vivo macrophage-to-feces RCT assay was performed
essentially as described (9). Briefly, thioglycollate-elicited mouse peritoneal macrophages were
harvested from donor mice, plated in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA)
supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA) and 1%
penicillin/streptomycin (Invitrogen), and allowed to adhere for 4h at 37°C under 5% CO 2 humidified air.
Macrophages were loaded for 24h with 50 μg/mL acetylated LDL and 3 μCi/mL 3H-cholesterol (Perkin
Elmer, Boston, MA, USA), followed by equilibration for 18h in RPMI 1640 medium containing 1%
penicillin/streptomycin and 2% bovine serum albumin (Sigma, St. Louis, MO, USA). For in vivo RCT
two million macrophages were injected intraperitoneally. Blood samples were taken 6, 24 and 48h
after injection, feces were collected continuously for 48h and livers were harvested after 48h and
stored at -80°C until further analysis. Counts in plasma were assessed directly by liquid scintillation
counting (Packard 1600CA Tri-Carb, Packard, Meriden, CT, USA), whereas liver was solubilized
(Solvable, Packard, Meriden, CT, USA) before counting (9). Feces were collected, dried, weighed and
thoroughly ground. Fecal bile acid and neutral sterol fractions were separated (10) and counts in the
respective fractions were related to the total amount of feces produced over the whole experimental
period. All counts were expressed as a percentage of the administered tracer dose.
Statistical analysis. All results are presented as means  SEM. Comparison between groups were
made using the Mann-Whitney test for independent samples. Outcomes of p<0.05 were considered to
be significant, except for the analysis of gene expression by real-time quantitative PCR (Student‘s Ttest, p<0.01). Analyses were performed using GraphPad Prism 5 software.
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