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