% C h o le s te r o l E fflu x /h
7
6
5
4
1
0
0
50
100
150
200
250
r S O F P r e In c u b a tio n T im e , m in
Supplementary Figure 1: Effects of rSOF + HDL preincubation time on
cholesterol efflux from THP-1 macrophages. HDL (2 mg/mL) and rSOF (1 µg/mL)
were incubated for various times and then efflux to the incubation mixtures
containing HDL (50 µg/mL) was determined. These data show that increased
time of preincubation of HDL with rSOF increases the rates at which the products
accept cellular cholesterol.
3
[ H ] C h o le s te r o l, c p m
CERM
H D L + neo H D L
180
160
140
120
100
80
60
40
neo H D L
20
0
15
20
25
30
35
l u ti o n V o l u m e , m L
Supplementary Figure 2:1: ESEC
profiles of media after incubation of THP-1
macrophages with HDL (2 mg/mL) that had been treated for various times
with rSOF (1 µg/mL). white circles, 1 h; gray circles, 2 h; black circles, 3 h.
The upper two figures are offset from the abscissa for better viewing. These
data show that increasing the time of pre incubation of rSOF with HDL
increases the efflux of cholesterol to CERM and neo HDL.
Net % FC efflux (by mass)
7
6
HDL (50g/ml)
+ SOF (1g/ml)
5
4
3
2
1
0
TO-901317 (1mol/L) AcLDL (100g/ml)
-
+
+
-
+
+
Supplementary Figure 3: Effects of various incubations on the mass of
cholesterol efflux as labeled. HDL was preincubated with rSOF and then with
THP-1 macrophages.
Cellular and media cholesterol were determined
enzymatically (Wako).
TO-901317 (1mol/L) AcLDL (100g/ml)
-
+
+
-
+
+
TLP, 15min
+SOF, 15min
A
60
[3H] Cholesterol, cpm
40
20
0
B
400
TLP, 2hrs
+SOF, 2hrs
200
0
0
5
10
15
20
25
30
Fraction Number
Supplementary Figure 4: SEC of TLP (200 µg/mL) with (18 h) and
without pre treatment with rSOF followed by incubation with
[3H]cholesterol-labeled THP-1 macrophages. A. 15 min incubation; B. 2
hour incubation. TLP (–); TLP post rSOF (- -).
300
A: HDL
200
15 min
120 min
100
3
[ H]Cholesterol, cpm
0
B: HDL-SOF
300
200
100
0
400 C: Neo HDL
300
200
100
0
0
5
10
15
20
25
30
Fraction Numbers
Supplementary Figure 5: Effect of rSOF on the elution profile
of media after incubation with [3H]cholesterol labeled THP-1
macrophages. A. HDL; B. HDL and rSOF reaction mixture after
an 18 h preincubation. C. Isolated neo HDL. Incubation times
are as labeled.
[3H] Cholesterol, cpm
500
pre incubation 15 min.
pre incubation for 120 min.
400
CERM
HDL
300
neo HDL
200
100
0
15
20
25
30
35
Elution Volume
Supplementary Figure 6: SEC of apo B-depleted plasma after preincubation
with rSOF (1 µg/mL) for 15 and 120 min and followed by incubation with THP1 macrophages for 30 min. Note the appearance of the[3H]cholesterolcontaining CERM peak and the shift in the elution profile of HDL to that of neo
HDL in the longer term preincubation.
12
HDL
HDL + SOF
8
4
3
[ H]CE formed/30min
16
0
0.0
0.03
SOF (g/mL)
0.3
Supplementary Figure 72: Effect of rSOF dose on LCAT activity.
HDL (2 mg/mL) was preincubated with rSOF at different concentrations
and assayed for CE formation by LPDS (1mg/mL) for 30 min after a 30
min preincubation with buffer and 10% BSA in a total volume of 0.5ml.
These data show that increasing the dose of rSOF that is preincubated
with HDL increases the amount of esterification by LCAT.
+ HDL
+ Neo HDL
40
60
B
30
40
20
20
DL
o /H
?
ne
RM
DL
o /H
L
ne
LD
0
CE
RM
/V L
0
?
DL
10
CE
%CE
A
Supplementary Figure 8: Fraction of [3H]cholesterol radiolabel
occurring as [3H]CE in various lipoprotein subclasses. Lipoprotein
fractions in A and B correspond to the peaks in Figures 6 A and B
respectively.
Supplemental Table 1: Kinetic Parameters for Cholesterol Efflux from THP-1 Cells to Various Acceptors*
Vmax (%/h)
Km
Cateff = Vmax/Km
Cateff (+SOF)/
Cateff (Control)
Control
+SOF
Plasma†
8.1 ± 0.08 8.1 ± 0.08
Plasma‡
6.1 ± 0.03 7.0 ± 0.16
9.0 ± 1.8
7.4 ± 0.3
HDL§
7.7 ± 0.5
11. ± 0.7
HDL||
4.8 ± 0.4
5.9 ± 0.4
TLP
Control
2.6 ± 0.3 %
3.4 ± 0.03 %
165 ± 58 µg/mL
31 ± 7 µg/mL
74 ± 18 µg/mL
* Based on data of Figure 2.
†Unloaded Cells.
‡ Loaded Cells.
§ HDL compared to SOF-HDL reaction mixture.
|| HDL compared to isolated neo HDL.
+SOF
Control
+SOF
1.15 ± 0.04 %
3.1 %/% plasma
7.0 %/% plasma
2.3
1.3 ± 0.09 %
1.8 %/% plasma
5.4 %/% plasma
3.0
53 ± 6 µg/mL
0.055 %-mL/h-µg
0.14 %mL/h-µg
2.6
24.3 ± 5.3 µg/mL
0.25 %-mL/h-µg
0.46 %-mL/h-µg
1.8
39 ± 9 µg/mL
0.065 %-mL/h-µg
0.15 %-mL/h-µg
2.3