Supplementary Materials for - Science Advances · 2017-04-17 · Eunha Kim, Jiyoon Beon, Seulgi Lee, Seung Ju Park, Hyoungjoon Ahn, Min Gyu Kim, Jeong Eun Park, Wooseob Kim, Jae-Min

advances.sciencemag.org/cgi/content/full/3/4/e1602296/DC1

Supplementary Materials for

Inositol polyphosphate multikinase promotes Toll-like receptor–induced

inflammation by stabilizing TRAF6

Eunha Kim, Jiyoon Beon, Seulgi Lee, Seung Ju Park, Hyoungjoon Ahn, Min Gyu Kim, Jeong Eun Park,

Wooseob Kim, Jae-Min Yuk, Suk-Jo Kang, Seung-Hyo Lee, Eun-Kyeong Jo,

Rho Hyun Seong, Seyun Kim

Published 21 April 2017, Sci. Adv. 3, e1602296 (2017)

DOI: 10.1126/sciadv.1602296

This PDF file includes:

fig. S1. Generation of myeloid lineage-specific conditional IPMK-null mice.

fig. S2. Validation of myeloid lineage-specific conditional IPMK-null mice.

fig. S3. IPMK depletion in macrophages blunts TLR-dependent inflammatory

responses.

fig. S4. IPMK depletion in RAW 264.7 macrophages down-regulates TLR-

dependent inflammatory responses.

fig. S5. IPMK depletion in macrophages does not alter TLR3-dependent

inflammatory responses.

fig. S6. Upstream TLR signaling regulators are not altered in IPMK-depleted

macrophages.

fig. S7. Overexpression of IPMK reduces TRAF6 ubiquitination.

SUPPLEMENTARY FIGURES

fig. S1. Generation of myeloid lineage-specific conditional IPMK-null mice. (A) A schematic of

myeloid lineage-specific conditional IPMK-null mice generation. Exon 6 was floxed in the targeted allele

and excised in the conditional knockout allele. (B) Levels of IPMK mRNA and protein in IpmkWT and

IpmkΔMac BMDMs were measured by RT-qPCR and immunoblotting, respectively. (C) Levels of IPMK

mRNA and protein in peritoneal macrophages from IpmkWT and IpmkΔMac mice were measured by RT-

qPCR and immunoblotting, respectively. (D) Levels of IPMK mRNA in muscle, white adipose tissue, and

liver from IpmkWT and IpmkΔMac mice were measured by RT-qPCR. The data shown are representative of

at least three independent experiments, and are presented as means ± SE (n = 3). *P < 0.05, ***P < 0.001

(Student’s t test).

fig. S2. Validation of myeloid lineage-specific conditional IPMK-null mice. (A and B) Flow

cytometric analyses were performed using cells from the peritoneal cavity (PECs), mesenteric lymph

nodes (MLNs), and spleen isolated from IpmkWT and IpmkΔMac mice. The cells were gated on specific cell

surface markers for macrophages (A) and monocytes (B). IpmkWT littermates served as controls for

IpmkΔMac mice. Data are representative of three mice per group from two independent experiments.

fig. S3. IPMK depletion in macrophages blunts TLR-dependent inflammatory responses. As shown

in Fig. 2C, the phosphorylation of signaling molecules was analyzed by immunoblotting lysates of

BMDMs stimulated for 2 hours with 100 ng/ml LPS. Densitometric quantitation was performed, and the

results were normalized with respect to control levels. The data shown are presented as means ± SE (n =

3). **P < 0.01, ***P < 0.001 (Student’s t test).

fig. S4. IPMK depletion in RAW 264.7 macrophages down-regulates TLR-dependent inflammatory

responses. (A to D) RAW 264.7 macrophages transfected with scrambled RNA (scRNA) or IPMK-

specific siRNA (siIPMK). Experiment was performed 48 hours after transfection. (A) Validation of Ipmk

depletion in RAW 264.7 macrophages transfected with scrambled or siIPMK as measured by qPCR. (B)

Pro-inflammatory cytokines Il-1, Il-6, and Tnf mRNA expression was measured by RT-qPCR in

scRNA or siIPMK transfected RAW 264.7 macrophages 6 hours after stimulation with 100 ng/ml LPS.

(C) Secreted levels of the cytokines IL-1, IL-6, and TNF were measured in scRNA or siIPMK

transfected RAW 264.7 macrophage culture media by ELISA 6 hours after stimulation with 100 ng/ml

LPS. (D) Phosphorylation of signaling molecules was analyzed by immunoblotting lysates of scRNA or

siIPMK transfected RAW 264.7 macrophages stimulated for 2 hours with 100 ng/ml LPS. Data shown are

representative of at least three independent experiments (A–D), and are presented as means ± SE (n = 3).

*P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test).

fig. S5. IPMK depletion in macrophages does not alter TLR3-dependent inflammatory responses.

(A) mRNA levels of the pro-inflammatory cytokines Il-6, Tnf, and Ifn were quantified in BMDMs 6

hours after stimulation with 10 g/ml poly (I:C). (B) Secreted levels of the cytokines IL-6 and TNF in

BMDM culture media were measured by ELISA 6 hours after stimulation with 10 g/ml poly (I:C). (C)

Phosphorylation of signaling molecules in BMDMs stimulated for 2 hours with 10 g/ml poly (I:C) was

analyzed by immunoblotting. In all BMDMs studies, IpmkWT littermates served as controls for IpmkΔMac

mice. Data shown are representative of at least three independent experiments (A–C), and are presented

as means ± SE (n = 3). *P < 0.05 (Student’s t test).

fig. S6. Upstream TLR signaling regulators are not altered in IPMK-depleted macrophages. (A)

Expression of mRNAs for negative regulators of TLR signaling pathway were measured by RT-qPCR in

BMDMs 6 hours after stimulation with 100 ng/ml LPS. (B) Expression of mRNAs for upstream

regulators of the TLR pathway was analyzed in BMDMs stimulated for 6 hours with 100 ng/ml LPS. (C)

Protein levels of major upstream factors (i.e., TLR4, MyD88, TRIF) were measured in BMDMs 2 hours

after stimulation with 100 ng/ml LPS. In all BMDMs studies, IpmkWT littermates served as controls for

IpmkΔMac mice. Data shown are representative of three independent experiments (A–C), and are presented

as means ± SE (n = 3). Student’s t tests revealed no significant changes.

fig. S7. Overexpression of IPMK reduces TRAF6 ubiquitination. HEK293T cells were transiently co-

transfected with FLAG-TRAF6 plus mock, HA-WT ubiquitin, HA-K48 ubiquitin, or HA-K63 ubiquitin,

plus GST or GST-IPMK expression plasmids. At 48 hours post-transfection, the cells were lysed, the

lysates were boiled at 95°C for 15 min and subjected to immunoprecipitation with an anti-FLAG antibody,

and immunoblotting was performed with anti-FLAG, anti-GST, or anti-HA antibodies.