Pellino3 ubiquitinates RIP2 and mediates NOD2-induced signaling

and protective effects in colitis

Shuo Yang, Bingwei Wang, Fiachra Humphries, Ruaidhri Jackson, Marc E. Healy,

Ronan Bergin,Gabriella Aviello, Barry Hall, Deirdre McNamara, Trevor Darby, Aoife Quinlan,

Fergus Shanahan, Silvia Melgar, Padraic G. Fallon and Paul N. Moynagh

SUPPLEMENTARY FIGURES

Nature Immunology: doi:10.1038/ni.2669

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Supplementary Figure 1: Pellino3 positively regulates MDP-induced activation of NF-

B in HEK293 cells

(a) Quantitative PCR of PELI3 mRNA expression in HEK293T cells transfected with control

or Pellino3-specific siRNA. (b) Normalised NF-B-regulated firefly luciferase expression in

HEK293T cells co-transfected with control or Pellino3-specific siRNA and an expression

construct encoding NOD2 (25 ng) and stimulated with MDP (10 μg/ml) for 18h. (c, d)

Normalised NF-B-regulated firefly luciferase expression in HEK293T cells co-transfected

with indicated amounts of expression constructs encoding NOD2 and (c) Pellino3s or (d)

Pellino3l and stimulated with MDP (10 μg/ml) for 18h. Data are presented as the mean +/-

S.E.M. of three independent experiments and were subjected to paired Student's t test. *, p

< 0.05; **, p < 0.01; ***, p < 0.001.

Nature Immunology: doi:10.1038/ni.2669

Nature Immunology: doi:10.1038/ni.2669

Nature Immunology: doi:10.1038/ni.2669

Nature Immunology: doi:10.1038/ni.2669

Nature Immunology: doi:10.1038/ni.2669

Nature Immunology: doi:10.1038/ni.2669

Supplementary Figure 6: Pellino3 interacts with and promotes ubiquitination of RIP2 in a manner

independent of the CARD domain and kinase activity of RIP2.

(a) Schematic representation of RIP2 Indicating domain structure (kinase, intermediate (IM), CARD)

with numbers indicating amino acid residues. (b, c) Immunoblot (IB) analysis of Flag-tagged RIP2,

various RIP2 truncation mutants and myc-tagged Pellino3l in lysates (Input) and (b) immunoprecipitated

Pellino3l or (c) immunoprecipitated RIP2 samples from HEK293T cells previously transfected with

constructs encoding Flag-tagged RIP2, the indicated Flag-tagged RIP2 truncation mutants and

myc-tagged Pellino3l. β-actin was used as a loading control. (d, e) IB analysis of Flag-tagged RIP2,

Flag-tagged RIP2-K47R,myc-tagged Pellino3l and (e) HA-ubiquitin in lysates (Input) and

immunoprecipitated RIP2 samples from HEK293T cells previously transfected with constructs encoding

Flag-tagged RIP2, Flag-tagged RIP2-K479R, myc-tagged Pellino3l and (e) HA-tagged ubiqutin .

(f) IB analysis of Flag-tagged RIP2, Flag-tagged RIP2-K209R and myc-tagged Pellino3l in lysates

(Input) and immunoprecipitated Pellino3-myc (left panel) or RIP2 (right panel) samples from HEK293T

cells previously transfected with constructs encoding Flag-tagged RIP2, Flag-tagged RIP2-K209R and

myc-tagged Pellino3l. β-actin was used as a loading control. (g) IB analysis of HA-ubiquitin,

Flag-tagged RIP2, Flag-tagged RIP2-K209R and myc-tagged Pellino3l in lysates (Input) and

immunoprecipitated RIP2 samples from HEK293T cells previously transfected with constructs

encoding Flag-tagged RIP2, Flag-tagged RIP2-K209R, myc-tagged Pellino3l and HA-tagged ubiqutin

(left panel) or HA-tagged ubiquitin (containing single K at residue 63 (K63a) (right panel).

Nature Immunology: doi:10.1038/ni.2669

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Supplementary Figure 7: The FHA and RING domains of Pellino3 are both required for

MDP-induced K63-linked polyubiquitination of RIP2 and downstream signalling in THP1 cells

(a) Immunoblot (IB) analysis of K63-linked ubiquitin and RIP2 in lysates (input) and

immunoprecipitated (IP) RIP2 samples and immunoblot analysis of phosphorylated (p-) and total

levels of IBa, p38, Jnk and Erk in lysates (input) from untreated or MDP (50 μg/ml)-treated

control or Pellino3 shRNA-knockdown THP1 cells, previously infected with an Plv empty vector (EV) or

one encoding wild-type Pellino3 long (Peli3l) or Pellino3 with a mutated FHA (Peli3lFm) or

RING (Peli3lRm) domain. (b-c) ELISA analysis of (b) IL-6 and (c) TNF protein expression in untreated

or MDP (50 μg/ml)-treated control or Pellino3 shRNA-knockdown THP1 cells previously infected with

an Plv empty vector or one encoding Peli3l, Peli3lFm or Peli3lRm. Data are presented as the

mean +/- S.E.M. of 3 independent experiments and were subjected to two-tailed Students t-test.

*, p < 0.05; ***, p < 0.001.

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Nature Immunology: doi:10.1038/ni.2669