Download Figure S1

Figure S1
+ p21siRNA
a
b
Ctrl siRNA
c
p21
β-actin
Figure S1. Knockdown of p21 in CD4+ T cells using siRNA. Expression of p21 was analyzed by RT-PCR 36
h post transfection. -actin was used as a house-keeping gene. P21-specific siRNA was performed using 8
nmol/ml (a), or 4nmol/ml (b) and siControl (c). This experiment is representative of repeated experiments using
cells from different donors.
Figure S2
+ Atv (0.25 μg/ml)
+ Atv (0.5 μg/ml)
+ Atv (1 μg/ml)
CD4
-Atv
Annexin V
Figure S2. Viability of CD4+ T cells following atorvastatin treatment. Representative dot plot from CD4+ T cells isolated from 1 HIV-1
seronegative individual in the absence of treatment or treated with atorvastatin (0.25 to 1 g/ml) for 4 days. Following treatment surface
staining was performed. Gated cells representing percentages of apoptotic CD4+ cells as measured by flow cytometry.
Figure S3
Negative
CD14
A
+ Atv (1 μg/ml)
+ Atv (0.5 μg/ml)
- Atv
p24
-
+
-
Atorvastatin
+
B
P21
β-actin
1
2
3
4
Figure S3. Atorvastatin treatment limits HIV-1 replication in CD14+ cells. (A) Representative flow cytometry dot plots
from CD14+ T cells in the absence or presence of atorvastatin (0.5 and 1g/ml) after infection with X-4-tropic tropic
HIV-1 isolates respectively. Data obtained by viral p24 antigen intracellular staining on day 4 post infection. These plots
are representative of three repeat experiments using cells from different donors. (B) RT-PCR reflecting -actin and p21
gene expression in monocyte-derived macrophages treated with atorvastatin (1 g/ml) for 48 h compared with
untreated cells prior to total RNA isolation.
P < 0.0001
100
1.0
80
60
40
B
20
0
0.5 mg/ml
1 mg/ml
R5-tropic HIV-1
0.5 mg/ml
%CD4+p24+
A
%p24 suppression
Figure S4
1 mg/ml
0.8
P < 0.0001
0.6
0.4
0.2
0.0
-Atv
+Atv (1µg/ml)
R5-tropic HIV-1
+ Atv (0.5 μg/ml)
X4-tropic R5-tropic
C
+ Atv (0.5 μg/ml)
+ Atv (1 μg/ml)
Uninfected
0
X4-tropic
D
p24
R5-tropic
CD4
p24
- Atv
+Atv (1µg/ml)
X4-tropic HIV-1
+ Atv (1 μg/ml)
CD4
- Atv
-Atv
X4-tropic HIV-1
Figure S4. Atorvastatin reduces HIV-1 infection in resting CD4+ T cells. (A) Percentages of p24 suppression in
CD4+ T cells that were exposed to atorvastatin (0.5-1g/ml) for 48 h post HIV-1 infection with R5 (HIV-1JR-CSF) and X-4tropic isolates. (B) Percentages of CD4+p24+ cells in non-activated CD4+ T cells in the presence of atorvastatin (1g/ml)
48 h after HIV-1 infection with R5 (HIV-1JR-CSF) and X-4-tropic (HIV-1LAI) isolates. Significance was tested using paired t
test. (C) Representative flow cytometry dot plots from CD4+ T cells infected and then treated with 0.5-1g/ml
atorvastatin post infection with X-4-tropic or R5-tropic HIV-1 isolates respectively. (D) Representative flow cytometry dot
plots from CD4+ T cells treated for 24 h with 0.5-1g/ml atorvastatin post infection with R5 (HIV-1JR-CSF) and X-4-tropic
(HIV-1LAI) isolates. CD4+ T cells from 5 HIV-1 seronegative donors were used for each viral isolate. Data are from 5
HIV-1 seronegative individuals infected with both X4-and R5-tropic viral isolates in vitro with duplicate cultures per
condition. Error bars indicate mean ± SEM from duplicate cell cultures from 5 HIV-1 seronegative individuals infected 4
separate times with both X4-and R5-tropic viral isolates in vitro. Each point represents an individual.
Figure S5
+Atv (1μg/ml)
siControl CD4
+Atv (0.5 μg/ml)
siRNAp21 CD4
R5-tropic HIV
-Atv
p24
Figure S5. Atorvastatin mediated upregulation of p21 reduces HIV-1 infection in activated CD4+ T cells.
Representative example of dot plots from activated CD4+ T cells infected in the presence of p21-specific or control siRNA
with R5-tropic HIV-1 Isolate in the presence (0.5 g/ml or 1 g/ml) or absence of atorvastatin. Viral infection was measured
by viral p24 antigen staining on CD4+ T cells using flow cytometry. These plots are representative of independent
experiments performed on three different donors for each viral isolate.
Figure S6
+ Atv (0.5 μg/ml)
+ Atv (1 μg/ml)
+ Mevalo.
+ Atv (0.5 μg/ml)
+ Mevalo. (1 mM)
+ Atv (1 μg/ml)
+ Mevalo. (1 mM)
X4-tropic (HIV-1LAI)
CD4
X4-tropic (HIV-1LAI)
- Atv
p24
Figure S6. Atorvastatin reduces infection of CD4+ T cells to HIV-1 infection and upregulates p21 through mevalonate
pathway.
Representative dot plots of non-activated CD4+ T cells in the presence of atorvastatin (0.5-1g/ml) for 48 h and also in the
presence or absence of L. mevalonate (1mM) post in vitro infection with X4-tropic viral isolate. Viral infection was measured
by viral p24 antigen staining on CD4+ T cells using flow cytometry. These plots are representative of independent
experiments performed on three different donors.