SUPPLEMENTARY DATA
Material and Methods
Expression vectors and transfections - The pCMV-LDLR expression plasmid was obtained
from Dr. Alan T. Remaley (NIH, Bethesda, MD) and used as template to generate the D206E,
V408M and D154N mutant LDLR expression plasmids using the Quickchange II Site-Directed
Mutagenesis Kit (Stratagene, La Jolla, CA). Plasmids were entirely sequence checked.
HEK293 cells stably expressing human PCSK9 (23) as well as naive HEK293 cells were
plated at a density of 106 cells on 6-well plates and transfected with wild type or mutant
pCMV-LDLR vectors using 0.5µg of DNA and 12.5µL of Effectene (Qiagen, Courtaboeuf,
France) per well. Cells were grown in DMEM containing 10% FCS for an additional 48h and
harvested prior to flow cytometry and western blot analyses.
Western blots - Cells were washed with cold PBS and harvested in modified RIPA buffer
containing Tris 0.05 M, NaCl 0.15 M, Na-deoxycholate 0.25%, Triton X100 1%, EDTA 1
mM, and 1 µg/mL of protease inhibitor cocktail (Sigma, Saint-Quentin Fallavier, France). The
mixture was centrifuged at 4000g for 5 min at 4°C to remove cell debris. Proteins (50µg)
were resolved on Nu-PAGE 3-8% gels in Tris-Acetate buffer (Invitrogen, Saint-Aubin,
France) under reducing conditions, transferred onto a Hybond-ECL nitrocellulose membrane
(GE Healthcare, Aulnay sous Bois, France). Membranes were probed with a chicken anti
human LDLR antibody (1:1000) (17)
-actin antibody (1:3000)
(Abcam, Cambridge, UK), using ECL plus (GE Healthcare), and analyzed using Quantity
One Analysis Software (BioRad, Marne la Coquette, France).
Supplementary Figure 1:
Comparative effects of wild-type rPCSK9 and rPCSK9-D374Y on LDLR cell surface
expression in primary skin fibroblasts. (A) Primary fibroblasts isolated from a
normolipidemic non-FH individual were grown in 0·5% FCS with 10g/mL mevastatin for
24h. Increasing doses of wild-type rPCSK9 (0, 1500, 3000, and 6000ng/mL) and of rPCSK9D374Y (0,150, 300, and 600ng/mL) was added to the culture medium for 4 hours prior to flow
cytometric analysis of cell surface LDLR expression. (B) Comparison between 6000ng/mL of
wild-type rPCSK9 and 600ng/mL of rPCSK9-D374Y on LDLR cell surface expression was
also performed in primary fibroblasts isolated from HeFH patients carrying either the D206E,
V408M, or D154N LDLR missense mutation. Histograms represent the mean ± SEM of 3
independent experiments. *p<0·05, **p<0·01 vs. the No-PCSK9 experimental condition.
Representative fluorescence charts are presented for each experimental condition.
Supplementary Figure 2:
Effect of PCSK9 on the expression of wild-type, D206E, V408M and D154N LDLR. HEK
293 cells stably expressing PCSK9 (in white) or not (in black) were transiently transfected
with wild-type LDLR (WT), D206E-LDLR, V408M-LDLR, or D154N-LDLR. (A) The
expression of the LDLR at the cell surface was assessed by flow cytometry. Representative
fluorescence charts are depicted below. (B) The whole cell expression of the LDLR was
assessed by western blot. Non-transfected (Non-Tx) cells were used as negative controls.
Histograms represent the mean±SEM of four independent experiments performed in
duplicate. *p<0.05 vs. 293 cells without PCSK9.
Supplementary Table :
Demographic and fasting plasma lipids values of FH patients recruited for primary lymphocyte analysis
Genotype Sex
D206E
D206E
D206E
D206E
D206E
D206E
D206E
V408M
V408M
V408M
V408M
V408M
V408M
V408M
D154N
D154N
D154N
D154N
D154N
M
F
M
M
M
F
F
F
M
M
F
M
M
F
F
F
F
M
F
Age
Treatment (mg/day)
CVD
70
51
66
68
78
59
58
57
50
38
44
19
19
49
47
60
48
61
70
Rosuvastatin 40mg
Atorvastatin 80mg
Rosuvastatin 40mg Ezetimibe 10mg
Rosuvastatin 40mg Ezetimibe 10mg
Atorvastatin 20mg
Rosuvastatin 40mg
Atorvastatin 80mg Ezetimibe 10mg
Atorvastatin 80mg
Rosuvastatin 40mg Ezetimibe 10mg
Rosuvastatin 40mg Ezetimibe 10mg
Atorvastatin 80mg
Atorvastatin 80mg
Atorvastatin 20mg Colesevelam 375mg
Rosuvastatin 20mg
Rosuvastatin 40mg Ezetimibe 10mg
Atorvastatin 40mg
Atorvastatin 80mg
Atorvastatin 80mg
Atorvastatin 80mg
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Non Rx Lipids (mmol/L)
TC
TG HDLC LDLC
Rx Lipids (mmol/L)
TC
TG HDLC LDLC
8·6
10·9
11
8·8
11·2
8·6
9·2
11·8
12·5
8
9·1
9·8
10
9·6
9
7·2
6·5
11·1
6·8
7·9
5
6·3
6·1
5·8
4·5
6·9
4·4
6
7
6·9
4·8
5·9
7·5
5·1
6·2
7·3
6
1
1·6
2·5
1·7
1·7
0·3
1
2·2
2
5·5
1·5
0·9
0·9
1·4
0·8
0·6
0·6
6·8
Non Rx Lipids: measured at time of diagnostic (without treatment)
Rx Lipids: latest laboratory results available (on lipid lowering medication)
1·4
1·1
1·2
0·9
1·2
1·7
0·8
1·3
1·3
0·7
0·9
1·3
1·1
1·1
1·4
1·3
0·9
0·9
6·7
9·1
8·6
7
9·2
6·7
7·9
9·5
10·3
7·5
8·1
8·5
7·9
7·2
5·6
5·3
6·8
1·5
3·3
1·4
1·2
1·1
0·6
0·6
2
0·9
3·8
1·4
1
0·7
1·8
1·2
0·8
0·5
0·6
1·4
1·3
0·9
1·3
1·2
1·5
1·9
1·1
0·8
0·9
0·8
1
1·1
1·1
1·1
2
1·1
1·1
1·1
1·2
5
5·5
3·1
4·5
4
3·7
3·3
5·2
3·3
3·5
5·4
5·3
3·4
4
4·8
3·5
4·8
5·9
4·1