Supplementary Figures legends
Figure-s 1. Expression of CD44 protein on 293T cells transfected with
CD44. Staining for CD44 (brown color) in (A) 293T cells transfected with
CD44 expression vector or (B) 293T cells transfected with empty vector
(pcDNA3.1). 293T cells were plated into a 10 cm dish the day before
transfection. 10µg of plasmid of either CD44 expression vector or empty
vector were transfected using FuGENE®HD Transfection reagent
(Roche, Indianapolis,IN) according to the manufacturer’s instructions.
Cells
were
fixed
at
three
days
post-transfection
with
2%
formaldehyde(Sigma, St. Louis, MO) and incubated with anti-human
CD44
antibody(R&D
systems,
Mineapolis,
MN)
followed
by
anti-mouse-IgG-peroxidase. Final development and staining was with
Sigma FAST 3, 3-diaminobenzidine tablets (Sigma, St. Louis, MO).
Figure-s 2. Expressed CD44 protein can bind to HA. Binding of HA to
CD44. (A) Transfection with CD44 expression vector; (B) transfection
with empty vector (pcDNA3.1). 293T cells were harvested at day three
post-transfection (from supplementary figure 1), washed with PBS twice,
incubated with fluorescein conjugated Hyalronan (FI-HA) (Sigma, St.
Louis, MO) for 30 minutes, and washed three times with PBS. 50,000
cells per condition were evaluated by flow cytometry (FACS).
1
Closed
histogram: cells without FI-HA incubation; open histogram: cells with
FI-HA incubation.
Figure-s 3. CD44 is on virion of HIVCD44. 10 ml of each Supernatant from
three conditions of 293T cells, all transfected with same amount of
plasmid: CD44 expression vector with empty vector (pcDNA3.1) (CD44),
empty vector (pcDNA3.1) with HIV plasmid of pNL4-3(HIVmock), and
CD44 expression vector with HIV plasmid of pNL4-3(HIVCD44) were
concentrated by 6% of Iodixanol solution gradients method 30, which can
pellet HIV particles without pellet proteins from supernatant, and the
pellet was resuspended in 1ml of PBS. CD44 were measured by Human
sCD44std instant ELISA kit (eBioscience, San Diego CA) according the
manufacture. Filled bar is supernatant from 293T cells, and open bar is
from pellet. (All data are mean ±SEM of duplicate each samples).
Figure-s 4. HIVCD44 has higher infectivity than HIVmock in M7-Lue cell line .
M7-Lue cells were inoculated with the same amount of p24 from both
293T cells made HIV stocks, HIV contained CD44 (HIVCD44), HIV without
CD44(HIVmock) in 10% FCS for three days. HIV infectivity was
measured by Bright-GloTM Luciferase assay system (Promega, Madison,
WI) and expressed as relative light units (RLU). (All data are mean ±SEM
of
triplicate
samples
and
representative
2
of
three
independent
experiments).
Figure-s 5. HIVCD44 has higher infectivity than HIVmock on primary CD4 T
cells. Primary stimulated CD4 T cells were inoculated with the same
amount of p24 from both 293T cells made HIV stocks, HIV contained
CD44 (HIVCD44), HIV without CD44(HIVmock) in 10% FCS for five hours,
then removed the supernatant, wash the cells three times with RPMI,
then cultured in completed RPMI+20u of IL-2. HIV infectivity was
measured by HIV-1 p24 production in supernatant at day seven
post-infection. Results are from duplicate assays of independent
experiments representative of at least three separate donors.
Figure-s 6. Both HIVCD44 and HIV
PBMC
infectivity respond to anti-CD44
beads in the M7-lue cell assay. Anti-CD44 beads (Mitenyibiotec Auburn,
CA) can enhance HIVPBMC infectivity
27
. We used the anti-CD44 beads in
the M7-Lue cell assay to compare HIV viral stocks of HIVCD44, HIVmock and
HIVPBMC.
M7-Lue cells were inoculated with the same amount of p24
from different HIV stocks, without (filled bar) or with (open bar) anti-CD44
beads for three days. HIV infectivity was measured by Bright-GloTM
Luciferase assay system (Promega, Madison, WI) and expressed as
relative light units (RLU). Anti-CD44 beads significantly enhanced both
HIVPBMC (p= 0.00012676) or HIVCD44 (p= 0.00039612) infectivity. However,
3
treatment with anti-CD44 beads did not affect infectivity of HIVmock (p=
0.92404636). (All data are mean ±SEM of triplicate samples and
representative of 3 independent experiments).
Figure-s 7. Removing of Fetal calf serum (FCS) during the the cells
inoculated with HIV reduces infectivity of both HIVCD44 and HIVmock in the
M7-lue cell assay. M7-Lue cells were inoculated with the same amount of
p24 of HIV stocks that made from 293T cells, without FCS (filled bar) or
with 10% of FCS (open bar) for five hours, then removed supernatant,
and washed the cells three time, and cultured in completed RPMI for
three days . HIV infectivity was measured by Bright-GloTM Luciferase
assay system (Promega, Madison, WI) and expressed as relative light
units (RLU). Infectivity of HIVCD44 is significantly higher in 10% of FCS
condition (p=0.00078315), however the significant is lost in FCS free
condition (p=0.0806). (All data are mean ±SEM of triplicate samples and
representative of 3 independent experiments).
Figure-s 8. HA reduced HIVCD44 infectivity on CD44 expressed Jurkat
cells. Flow cytometry (FACS) analysis of Jurkat cells at day seven of
transfected with CD44 or empty vector and infected with either HIVCD44 or
HIVmock for three days. Cells were permeated intracellular staining of
HIV-p24 (KC-57RD1-PE) (Beckman coulter) and CD44 (hCD44-APC) (R&D
4
system), and 50,000 of each stained cells were measured by
EasyCyte6HT-2L (Millipore, MA), (No-HIV) Juarkat cells with transfection
of either CD44 expression vector or empty vector without HIV infection;
(HIVCD44) cells were infected by HIVCD44, and (HIVmock) cells were infected
by HIVmock. (A) is CD44 expression vector transfected Jurkat cells, and
(B) is empty vector transfected Jurkat cells. (All data are mean ±SEM
from duplicate samples and two experiments).
Figure-s 9. The 12 images of Z-stack projections from laser scanning
microscopy (LSM) analysis of Figure 4A: HA on CD4+ T cell without
hyaluronidase treatment. Images are 40x; HA-staining-Alexa488 (blue);
cell membrane staining-Texas Red (red) (Life Technologies, Carlsbad,
CA); and nuclear staining-DAPI (white) (Life Technologies, Carlsbad, CA).
The Z-stack projections started at number 1 and finished at number 12.
Figure-s 10. 100µg of HA or 1µM of Gö6976 have no impact on primary
unstimulated CD4+ T cell number or viability. (A) Total cell number from
each condition. (B) % viability of cells for each condition. Primary
unstimulated CD4+ T cells were treated with either 100µg of HA(Sigma,
St. Louis, MO), 1µM of Gö6976(Sigma, St. Louis, MO), or 1µl of DMSO
(Sigma, St. Louis, MO) and medium for five hours, then the Guava
ViaCount assay (Millipore, Hayward CA) was used to measure total cell
5
number and viability. There are no significant differences between
conditions.
Results
are
from
duplicate
assays
of
independent
experiments representative of at least three separate donors.
Figure-s 11. Both high and low molecular weight exogenous HA inhibited
HIVPBMC infection. M7-Lue cell assays.
HHA: high molecular weight
exogenous HA, >950KDa(R&D systems, Mineapolis, MN); LHA: low
molecular weight exogenous HA, ≈29 KDa(R&D systems, Mineapolis,
MN); and HA: mixed molecular weight exogenous HA (Sigma, St. Louis,
MO). RLU (relative light units) is a measurement for luciferase activity.
All exogenous HA forms decreased HIVPBMC infectivity at day three postinfection. (Data are mean ±SEM of triplicate samples and representative
of three independent experiments).
Figure-s 12. HIV did not grow in unstimulated CD4+T cells unless
stimulated by PHA. During Figure 3B assays, after HIV infection, the
unstimulated CD4+ T cells were divided into two parts, one with PHA
(Roche, Indianapolis, IN) stimulation for 24 hours, and one without PHA
stimulation. HIV production was detected on day 7 post-infection in the
PHA-stimulated cells, but was not detected in the unstimulated cells at
day 15 post-infection. (A) CD4+T infected with HIVCD44; and (B) CD4+T
infected with HIVmock (open bar is stimulated with PHA, filled bar is
6
without PHA stimulation). Hase: Hyaluronidase (Sigma, St. Louis, MO)
treatment; medium: medium treatment; Hase+HA: Hyaluronidase
treatment with 100µg of exogenous HA; Medium+HA: medium treatment
with100µg of exogenous HA. (All data are mean ±SEM from duplicate
samples and representative of at least three donors).
Figure-s 13.
Exogenous HA inhibited HIVCD44 infectivity on PHA
stimulated CD4 T cells. CD4 T cells were stimulated with 3µg of PHA-M
(Roche, Indianapolis, IN) and 20 units of hIL-2(Roche, Indianapolis, IN)
for three days, washed once with FCS-free RPMI, treated with or without
100µg of exogenous HA (Sigma, St. Louis, MO) for one hour, inoculated
with 1000pg HIV-p24 of HIVCD44 or HIVmock for five hours, washed three
times with complete RPMI, and resuspended in complete RPMI with 20
units of hIL-2. HIV infectivity was measured by HIV-1 p24 production in
supernatant at day seven post-infection. (All data are mean ±SEM from
duplicate samples and representative of at least three donors).
7