Nature Immunology doi: /ni.3268

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2 Supplementary Figure 1 Loss of Mst1 and Mst2 increases susceptibility to bacterial sepsis. (a) H&E staining of colon and kidney sections from wild type and Mst1 -/- Mst2 fl/fl Vav-Cre mice. Scale bar, 50 µm. (b, c) Representative spleen tissues: spleen weight (b) and H&E staining of spleen sections (c) from wild type and Mst1 -/- Mst2 fl/fl Vav-Cre mice. Scale bar, 100 µm. (d) White blood cell (WBC), circulating lymphocyte (LYM), monocyte (MON) and granulocyte (GRA) counts in Mst1 fl/fl Mst2 fl/fl (WT) and Mst1 fl/fl Mst2 fl/fl Lyz2-Cre (cdko) mice. Each dot represents an individual mouse, and horizontal bars indicate the mean. (e) Flow cytometric analysis of Gr-1 + CD11b + neutrophil and F4/80 + CD11b + macrophage populations in the bone marrow and spleens of WT and cdko mice, as determined with anti-gr-1, anti-f4/80 and anti-cd11b antibodies. (f) Flow cytometric analysis of B220 + B cell, CD3 + T cell, the naïve T cell (CD62L high CD44 low ) and the effector T cell (CD62L low CD44 high ) populations in the spleen, lymph node (LN) or blood of WT and cdko mice, with the indicated antibodies. (g) Flow cytometric analysis of Gr-1 + CD11b + neutrophil and F4/80 + CD11b + macrophage populations in blood of WT and cdko mice subjected to cecal ligation and puncture (CLP) for 24h. (h) H&E staining of kidney sections from WT or cdko mice subjected to sublethal CLP. Scale bar, 50 µm. (i) WT BMDMs were stimulated with different TLR agonists for the indicated times, followed by immunoblot analysis with the indicated antibodies. Data were assessed with Student s t-test and are represented as mean ± s.d. ns, not significant, * p<0.05, *** p<0.001 compared with respective controls or as indicated (b, n=10; d, n=8; e, n=3; g, n=4). Data are representative of three independent experiments

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4 Supplementary Figure 2 Mst1- and Mst2-deficient myeloid cells are defective in killing bacteria and induction of mros. (a) Mst1 fl/fl Mst2 fl/fl (WT) and Mst1 fl/fl Mst2 fl/fl Lyz2-Cre (cdko) BMDMs or neutrophils were treated with indicated TLR agonists for 6 h. LPS, Lipopolysaccharide; Pam3, Pam3CSK4; LTA, Lipoteichoic acid; PIC, poly(i:c). mros (MitoSox) and total cellular ROS (CM-H2DCFDA) production were measured by flow cytometry. (b) Luminometry of ROS production by WT and cdko BMDMs or neutrophils infected with E. coli (MOI, 10) or L. monocytogenes (Lm, MOI, 10). RU, relative units. Results are represented as mean ± s.d. n=3. (c) WT and cdko BMDMs or neutrophils were treated with antimycin A, oligomysin or rotenone for 6 h. mros production was measured as described in (a). (d-f) Representative transmission electron micrographs (d, Scale bar, 0.5 µm), the number of mitochondria (d), western blots of the major electron transport chain components (e) and the mitochondrial potential shown by JC-1 staining (f, Scale bar, 50 µm) of DMDMs from WT and cdko mice. Data were assessed with Student s t test and are represented as mean ± s.d. (d, n=20). ns, not significant. Data are representative of three independent experiments.

5 Supplementary Figure 3 Treatment with cytochalasin D diminishes the induction of mros and cellular ROS in BMDMs after stimulation with LPS. (a) Mst1 fl/fl Mst2 fl/fl (WT), Mst1 fl/fl Mst2 fl/fl Lyz2-Cre (cdko), Rac1 G12V and cdko-rac1 G12V BMDMs were treated with the indicated doses of cytochalasin D and were immunostained with F-actin antibodies (green). Scale bar, 20 µm. (b) WT BMDMs were pretreated with the indicated doses of cytochalasin D for 1 h followed by LPS stimulation for 3 h. mros and cellular ROS production were measured by staining cells with MitoSOX and

6 CM-H2DCFDA for 30 min, respectively, followed by flow cytometry. Data are representative of three independent experiments.

7 Supplementary Figure 4 Interaction of Mst2 and PKC isoforms. (a) The interaction between Mst2 and PKC kinases. Co-immunoprecipitation assay of 293T cells expressing HA-Mst2 and Flag-tagged PKCα, β, γ, δ, ε, η, θ, ι or ζ as indicated. Cell lysates were immunoprecipitated with anti-flag antibody and analysed by immunoblotting with antibodies as indicated. (b) Mst2 binds the N-terminal regulatory domain of PKCα. Co-immunoprecipitation assay of 293T cells

8 expressing HA-Mst2 and Flag-PKCα N-terminal (NT) or Flag-PKCα C-terminal (CT) as indicated combination. The cell lysates and immunoprecipitates were analysed by immunoblotting with antibodies as indicated. (c) Phosphorylation sites of PKCα were detected by mass spectrometry analysis of bacterially expressed GSTtagged PKCα(NT) from an in vitro kinase reaction with triple HA-tagged Mst2 immunoprecipitated from HeLa cells. Data are representative of three independent experiments.

9 Supplementary Figure 5 TRAF6 activity is essential for the K63-linked ubiquitylation of Rac1. (a) The distribution of Rac1 (red) in Mst1 fl/fl Mst2 fl/fl (WT) BMDMs, Mst1 fl/fl Mst2 fl/fl Lyz2-Cre (cdko) BMDMs and WT BMDMs treated with NSC23766 after phagocytosis with GFP-E. coli. Images shown are representative of approximately 100 cells. Scale bar, 20 µm. (b) Pull-down experiments were performed using purified Flag-Rac1 WT, Flag-Rac1 G12V or Flag-Rac T17N

10 incubated with lysates from LPS-treated BMDMs. Immunoblots show the association of TRAF6 with Flag- Rac T17N. (c) Ubiquitination and immunoprecipitation assay of 293T cells transfected with plasmids expressing Flag- Rac1, Myc-TRAF6 and HA-ubiquitin (Ub), HA-ubiquitin K48 (Ub 48) or HA-ubiquitin K63 (Ub 63). Cell lysates were immunoprecipitated with anti-flag antibody. The cell lysates and immunoprecipitates were analysed by immunoblotting with antibodies as indicated. Anti-HA immunoblotting shows the level of Rac ubiquitylation. (d) Knock-down of TRAF6 expression levels in BMDMs or RAW264.7 cells with different TRAF6 shrnas. (e) The ubiquitination levels of WT or mutated Rac1 with the indicated substitutions of lysine (K) with arginine (R) in 293T cells. Data are representative of three independent experiments.

11 Supplementary Figure 6 TRAF6 maintains a Rac1 activation state via K63-linked ubiquitination. (a) HeLa cells were transfected with plasmids expressing Rac1 WT, Rac1 G12V or Rac1 TN17, and/or TRAF6 as indicated combination. Confocal microscopy shows the co-localization of TRAF6 (green) with Rac1 TN17 (red),

12 but not with Rac1 G12V (red), at the cell periphery. Images shown are representative of approximately 100 cells. Scale bar, 20 µm. (b) Ubiquitination and immunoprecipitation assay of 293T cells expressing Flag-Rac1, Myc-TRAF6 and/or HA-Ub and treated with or without NSC23766 as indicated. Cell lysates were immunoprecipitated with anti- Flag antibody and analysed by immunoblotting with antibodies as indicated. Anti-HA immunoblotting shows that Rac ubiquitination by TRAF6 is reduced by the Rac1 inhibitor. Data are representative of three independent experiments.

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14 Supplementary Figure 7 Rac1 G12V enhances assembly of the TRAF6-ECSIT complex. (a-c) ECIST or Rac1 binds to the math domain of TRAF6. Co-immunoprecipitation assay of 293T cells expressing HA-ECSIT (a) or HA-Rac1 T17N (b), and Flag-TRAF6 full-length (FL) or fragments as indicated. Cell lysates were immunoprecipitated with anti-flag antibody and analysed by immunoblotting with antibodies as indicated. Schematic representation of the domain organization of TRAF6 is shown (c). (d) Loss of the K63-linked ubiquitination of inactivated Rac2 D57N by TRAF6 in 293T cells. Ubiquitination and co-immunoprecipitation assay of 293T cells expressing Flag-Rac2 WT, Flag-Rac2 D57N and/or Myc-TRAF6, plus WT ubiquitin (HA-Ub) or K63-linked ubiquitin ([63 only]ha-ub) as indicated. (e) HeLa cells were transfected with expression plasmids for Rac1 WT, Rac1 G12V or Rac1 TN17. Confocal microscopy shows enhanced co-localization of TRAF6 (green) with ECSIT (red) mediated by active Rac1 G12V (purple). Images shown are representative of approximately 100 cells. Scale bar, 20 µm. Data are representative of three independent experiments.

15 Supplementary Figure 8 Mst1 and Mst2 positively regulate the induction of phagocyte ROS and bactericidal activity. (a) A proposed working model for TLR-Hippo signalling-mediated ROS production. During an antimicrobial response, Toll-like receptor (TLR) signalling triggers the Mst1 and Mst2 kinases to directly phosphorylate PKCα at Ser 226 and Thr 228, leading to its activation, which promotes the dissociation of the GDPdissociation inhibitor LyGDI from Rac and enhances the GTP charging of Rac. Activated Rac then triggers the assembly of the TRAF6-ECSIT complex, controlling mitochondrial trafficking and promoting mitochondrionphagosome juxtaposition and thereby augmenting mitochondrial ROS (mros) production. TRAF6 further mediates the Lys (63)-linked ubiquitination of active Rac, maintaining the GTP charging of Rac and the subsequent activation of ROS-generating machinery during an antimicrobial response.