anti-
Gr1
104
103
102
101
100
anti-F4/80100 101 102 103 104
WT TRPV2KO
anti-
Gr1
104
103
102
101
100
anti-F4/80100 101 102 103 104
anti-
CD
11b
104
103
102
101
100
anti-F4/80100 101 102 103 104
anti-
CD
11b
104
103
102
101
100
anti-F4/80100 101 102 103 104
42.3 45.2
40 42.5
Link et al. Supplementary Figure 1
Macro Mono PMN Lymph
WT
KO
16
12
8
4
0
a
b WT Phalloidin KO Phalloidinc
Cel
lspe
rm
ouse
(10
5 )
Nature Immunology: doi:10.1038/ni.1842
WT KO KO +KCl
WT KO KO +KCl
1
0.8
0.6
0.4
0.2
0
Phagocytosis Binding
Control
Accutase
Inde
x(a
rbitr
ary
units
)
P = 0.01
P = 0.0002
P = 0.001
P = 4 x 10-5
Link et al. Supplementary Fig. 2
Nature Immunology: doi:10.1038/ni.1842
WT KO KO+KCl
WT KO KO+KCl0
0.6
0.4
0.2
Bin
ding
Inde
x
Link et al. Supplementary Figure 3
a b
†
Nature Immunology: doi:10.1038/ni.1842
Untreated U73122 PiceatannolPKC ζ
pseudo-substrateCalphostin C Akt inhibitor X Wortmannin PP2
α-T
RP
V2
Brig
htfie
ld
Link et al. Supplementary Fig. 4
Nature Immunology: doi:10.1038/ni.1842
anti-CD16/32 anti-CD64
anti-CD11b anti-CD18
Nor
mal
ized
prev
alen
ce
Isotype control WT TRPV2 KO
Link et al. Supplementary Figure 5
Nature Immunology: doi:10.1038/ni.1842
KCl +3m3FBS
KCl +adenosine
KCl +no drug
KCl +wortmanninuntreated
ba
WT
KO
untreated IC
Link et al. Supplementary Figure 6
Nature Immunology: doi:10.1038/ni.1842
SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1: Flow cytometric analysis of peritoneal macrophages. (a)
Scatter plots from flow cytometric examination of resident peritoneal cells from a
representative wild-type and TRPV2KO mouse pair. Percent macrophages are indicated
in gated regions. b. Quantification of cell types in peritoneal lavage samples from each
genotype. Mean ± SEM; wild-type, n = 10; TRPV2KO, n = 9. Macrophages were
defined as F4/80 and CD11b high/Gr1, CD3, and B220 negative. Lymphocytes were
defined as CD3 or B220 positive. Neutrophils were defined as Gr1 high/ CD11b medium.
c. Actin staining of wild-type and TRPV2KO peritoneal macrophages with rhodamine-
phalloidin. Scale bar, 20 µm.
Supplementary Figure 2: Discrimination between internalized versus noninternalized
particles during phagocytosis and binding. Following phagocytosis (5 min) or binding (5
min) in the presence of 10 µM cytochalasin D of IgG-coated latex beads, binding or
phagocytosis index was quantified in wild-type, TRPV2KO, or KCl-treated TRPV2KO
macrophages before (black bars) and after (open bars) incubation with Accutase (100%,
Sigma, 15 min, 37°C) to remove noninternalized particles. Mean ± SEM, n = 3 wells per
condition per genotype. Note that Accutase treatment removes nearly all particles
incubated under binding conditions, but spares most particles incubated under
phagocytosis conditions in wild-type mice or in TRPV2KO cells treated with KCl. There
is some reduction in particles associated with TRPV2KO cells even under phagocytosis
conditions, suggesting that not all phagosomes had closed.
Nature Immunology: doi:10.1038/ni.1842
Supplementary Figure 3: Defective phagocytosis in TRPV2 deficient BMM and rescue
by KCl. a. Representative photomicrographs of wild-type, TRPV2KO, and KCl-treated
TRPV2KO BMMs following 5 min phagocytosis of IgG-coated latex beads (2 µm, left).
Wild-type and TRPV2KO photos show cells exposed to beads under control conditions.
KO + KCl, TRPV2KO cells were exposed to beads with KCl (50 mM) added to the
medium. b. Corresponding phagocytic indices. Mean ± SEM, n = 3 mice per genotype,
each assayed in duplicate. † P < 10-4.
Supplementary Figure 4: Pharmacological inhibition of TRPV2 phagosomal
recruitment. Wild-type macrophages are shown after 5 min phagocytosis of IgG-coated
beads (arrowheads), without drugs or in the presence of PLC inhibitor U73122 (10 µM),
Syk kinase inhibitor piceatannol (20 µM), PKC ζ pseudosubstrate (40 µM), gernal PKC
inhibitor calphostin C (500 nM), Akt inhibitor X (10 µM), PI3 kinase inhibitor
wortmannin (100 nM), or Src kinase inhibitor PP2 (1 µM). Top, TRPV2
immunofluorescence. Bottom, brightfield images. Scale bar, 8 µm.
Supplementary Figure 5: Phagocyte receptor expression levels are similar between
wildtype and TRPV2 deficient macrophages. Representative histograms from flow
cytometric data of wild-type macrophages (F4/80 positive, CD11b positive, CD3
negative, B220 negative) in acutely isolated peritoneal lavage. Histograms compare
wild-type (blue traces) and TRPV2KO (red traces) macrophages and isotype controls
(green traces), with respect to intensity of surface binding by antibodies against Fcγ
Nature Immunology: doi:10.1038/ni.1842
receptors (anti-CD16/32, anti-CD64) (6) and complement C3 receptor (anti-CD11b, anti-
CD18) (7).
Supplementary Figure 6: IC-induced actin depolymerization. a. Representative
photomicrographs of wild-type and TRPV2KO BMMs, stained with rhodamine-
phalloidin, which are untreated or following a 2 min stimulation with ICs at 37°C. b.
Representative photomicrographs of TRPV2KO BMMs, stained with rhodamine-
phalloidin, which are untreated, treated with KCl (50 mM), KCl + wortmannin (100 nM,
PI(3)K inhibitor), KCl + adenosine (300 µM, PI4 kinase inhibitor), or KCl + 3m3FBS
(10 µM, PLC activator). Scale bar, 10 µm.
Nature Immunology: doi:10.1038/ni.1842
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