SUPPLEMENTARY INFORMATION doi:10.1038/nature10632 Supplementary Figure 1 Lyn mediates neutrophil wound responses as a redox sensor. a, A schematic model. Wounded epithelial cells release H 2 O 2 by an oxidative burst 1. Wound-derived H 2 O 2 directly activates Lyn in neutrophils through oxidation of cysteine 466, leading to neutrophil recruitment to wounds. b, A proposed model of H 2 O 2 -mediated regulation of Lyn s dual positive and negative pathways. In this model, we propose that H 2 O 2 directly activates Lyn while inhibiting the redox-sensitive phosphatases (such as SHIP and SHP-1) comprising Lyn s inhibitory pathway 2. www.nature.com/nature 1
RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 2 SFKs mediate neutrophil wound responses. a, Immuno uorescence of psfk (phosphorylation of SFK activation loop tyrosine) by mab (D49G8) in Tg(mpx:mCherry) at 30 min after tail transection. mcherry labels neutrophils. b, Quanti cation of uorescence intensity of psfk in neutrophils (wound: 55 cells (7 larvae), no wound: 32 cells (8 larvae)). c, 2 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH Immuno uorescence of psfk in Tg(mpx:mCherry) with/without DPI at 30 min after tail transection. d, Quanti cation of uorescence intensity of psfk in neutrophils (DMSO: 41 cells (10 larvae), DPI: 41 cells (6 larvae)). e, Neutrophil recruitment to wounds and ns at 1h and 2h post wounding with/without PP2 (1h/ctrl: 25 larvae, 1h/PP2: 17 larvae, 2h/ctrl: 21 larvae, 2h/PP2: 19 larvae). f, Neutrophil recruitment to wounds and ns at 1 hpw with/without PP1 or PP2 (ctrl: 44 larvae, PP1: 24 larvae, PP2: 23 larvae). g, Tracking of spontaneous random motility of neutrophils in the cephalic mesenchyme with/without PP2. h, Quanti cation of 3D velocity of neutrophils during random motility (DMSO: 14 cells, PP2: 11 cells). *P<0.05, two-tailed unpaired t-test (b,d,e,h) and one-way ANOVA with Dunnett post test (f). Scale bars: 50 μm. www.nature.com/nature 3
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SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 3 SFKs mediate early neutrophil wound responses. a, Time-course analysis of neutrophil accumulation around wounds generated with a needle. 3 dpf larvae were treated with DMSO, DPI or PP2, wounded and xed at each time point, and neutrophils were stained with Sudan Black (1h/DMSO: 20 larvae, 2h/DMSO: 21 larvae, 4h/DMSO: 21 larvae, 1h/DPI: 20 larvae, 2h/DPI: 20 larvae, 4h/DPI: 18 larvae, 1h/PP2: 21 larvae, 2h/PP2: 20 larvae, 4h/PP2: 22 larvae). b, Time-course analysis of neutrophil accumulation around wounds generated by tail transection with a razor blade. 3 dpf larvae were treated with DMSO, DPI or PP2, wounded and xed at each time point, and neutrophils were stained with Sudan Black (1h/DMSO: 20 larvae, 2h/DMSO: 20 larvae, 4h/DMSO: 21 larvae, 1h/DPI: 20 larvae, 2h/DPI: 21 larvae, 4h/DPI: 9 larvae, 1h/PP2: 20 larvae, 2h/PP2: 20 larvae, 4h/PP2: 20 larvae). c, 3 dpf larvae were wounded with a needle, treated with DMSO or PP2 at 1 hpw, and xed at 6 hpw. Neutrophils were stained with Sudan Black (DMSO: 23 larvae, PP2: 23 larvae). PP2 did not affect resolution of in ammation when added at 1 hpw. d, Time-lapse tracking of neutrophils photolabeled at wounds. 3 dpf Tg(mpx:Dendra2) 3 larvae were wounded with a razor blade. At 1 hpw, larvae were treated with DMSO or PP2 and neutrophils at wounds were photolabeled by photoconverting the uorescence of Dendra2 from green (488 nm) to red (543 nm) (white lines: photoconverted areas, green arrows: neutrophils which were not photolabeled, purple arrows: auto uorescence of pigment). Photolabeled neutrophils were tracked up to 9.5 hpw (DMSO: 10 larvae, PP2: 10 larvae, pooled analysis of two separate experiments). PP2 did not affect resolution of in ammation mediated by neutrophil reverse migration. Error bars indicate s.e.m. Scale bar: 50 μm. www.nature.com/nature 5
RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 4 Neutrophil responses to exogenous stimulants. a, Diagram of H 2 O 2 injection into the otic cavity of 3 dpf zebra sh. Neutrophils were stained 20 minutes after injection of 10 μm H 2 O 2. b, Representative pictures of Sudan Black staining with/without PP2. Generation of endogenous H 2 O 2 was prevented by DPI treatment. Blue circles outline the otic cavity. c, Neutrophil recruitment to the H 2 O 2 -injected otic cavity was inhibited by PP2 (DPI/Ctrl: 21 larvae, DPI/H 2 O 2 : 17 larvae, DPI+PP2/Ctrl: 17 larvae, DPI+PP2/H 2 O 2 : 18 larvae). 6 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH d, Diagram of bath application of 100 μm H 2 O 2 in combination with wounding in 3 dpf zebra sh. Neutrophils were stained 30 minutes post wounding. e, Representative pictures of Sudan Black staining with/without PP2. Generation of endogenous H 2 O 2 was prevented by DPI treatment. f, Neutrophil accumulation around wounds was inhibited by PP2 (DPI/Ctrl: 24 larvae, DPI/H 2 O 2 : 24 larvae, DPI+PP2/Ctrl: 24 larvae, DPI+PP2/H 2 O 2 : 24 larvae). g, Under-agarose assay using human neutrophils and 100 nm fmlp (n=3). Error bars indicate s.e.m. *P<0.05, one-way ANOVA with Bonferroni post-test (c,f) and two-tailed unpaired t-test (g). www.nature.com/nature 7
RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 5 Zebra sh Lyn. a, Tg(mpx:Dendra2) larvae at 3 dpf were used for ow cytometry. Dendra2-low (macrophages) and Dendra2-high (neutrophils) fractions are boxed (blue). Cells from wild-type larvae (red) were used to set the lowest level of expression. b, Alignment of amino acid sequences of human LYN and zebra sh Lyn. C224 and C466 were indicated with arrows. c, Neutrophil number in the CHT of 2.5 dpf larvae with/without lyn morpholinos (ctrl: 25 larvae, lyn MO1: 23 larvae, lyn MO2: 25 larvae). d, Representative pictures of Sudan Black staining of neutrophils in the CHT. *P<0.05, one-way ANOVA with Dunnett post test (c). 8 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 6 Zebra sh macrophages. a, 2.5 dpf Tg(mpx:Dendra2) larvae injected with lyn MO or buffer were treated www.nature.com/nature 9
RESEARCH SUPPLEMENTARY INFORMATION with DMSO, 20 μm PP2 or 100 μm DPI, wounded and xed 30 minutes post wounding. The xed larvae were immunostained with L-Plastin antibody, which detects leukocytes. The numbers of neutrophils (Dendra2 positive and L-Plastin positive cells) and macrophages (Dendra2 negative and L-Plastin positive cells) at wounds were quanti ed. b, Representative immuno uorescence pictures. *P<0.05, one-way ANOVA with Dunnett post test. Scale bar: 50 μm. 10 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH www.nature.com/nature 11
RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 7 Lyn knockdown inhibits neutrophil responses to H 2 O 2 but not to LTB 4. a, 2.5 dpf larvae injected with lyn MO or buffer were bathed in 100 μm H 2 O 2 following wounding. Generation of endogenous H 2 O 2 was prevented by DPI. The larvae were xed 30 minutes post wounding and stained with Sudan Black. Lyn knockdown inhibits neutrophil responses to exogenous H 2 O 2 (Ctrl/Ctrl: 19 larvae, Ctrl/H 2 O 2 : 20 larvae, lyn MO/ctrl: 20 larvae, lyn MO/H 2 O 2 : 19 larvae). b, Representative pictures of Sudan Black staining of 2.5 dpf larvae. c, 2.5 dpf larvae were bathed in 30 μm LTB 4 for 30 minutes, xed and stained with Sudan Black (DMSO: 21 larvae, DPI: 20 larvae). d, Representative pictures of Sudan Black staining of 2.5 dpf larvae bathed in 30 μm LTB 4. Neutrophils in the outlined ventral n were counted for quanti cation. e, 2.5 dpf larvae injected with lyn MO or buffer were bathed in 30 μm LTB 4 for 30 minutes, xed and stained with Sudan Black (ctrl: 66 larvae, lyn MO: 61 larvae). Lyn knockdown increased the number of neutrophils localized in the n upon LTB 4 stimulation. This increase correlates with the increase in the total number of neutrophils in the CHT of lyn morphants (Supplementary Fig.5c, d). f, Representative pictures of Sudan Black staining. *P<0.05, one-way ANOVA with Bonferroni post-test (a) and two-tailed unpaired t-test (c,e). 12 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 8 Cysteine 466 in zebra sh Lyn. a, C466 is located at the C-terminus of the kinase domain and is phylogenetically conserved. b, BIAM-labeled Lyn WT and C466A detected by streptavidin-conjugated dye. c, Quanti cation of b (n=3). d, Western blot of MAPKs in HEK293 cells expressing Lyn ectopically. e, Western blot of ERK phosphorylation in HEK293 cells expressing Lyn WT and C466A. f, Quanti cation of e (n=4). Error bars indicate s.e.m. *P<0.05, paired t-test. www.nature.com/nature 13
RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 9 Cysteine 468 in human LYN. a, Western blot of human LYN and ERK in HEK293 cells expressing human LYN ectopically. b, Western blot of autophosphorylation of LYN and phosphorylation of ERK in HEK293 cells expressing human LYN WT and C468A. c, Quanti cation of b (n=3). d, BIAM-labeled human LYN WT and C468A detected by streptavidin-conjugated dye. e, Quanti cation of d (n=3). Error bars indicate s.e.m. *P<0.05, paired t-test. 14 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 10 Tg(mpx:lyn WT-GFP) and Tg(mpx:lyn C466A-GFP). a, Neutrophils in the CHT of Tg(mpx:lyn WT-GFP). b, Neutrophils in the CHT of Tg(mpx:lyn C466A-GFP). c, Punctate localization of Lyn(WT)-GFP in a neutrophil. d, Punctate localization of Lyn(C466A)-GFP in a neutrophil. Scale bars: 50 μm (a), 10 μm (c). www.nature.com/nature 15
RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 11 Lyn regulates Erk activity in neutrophils in vivo. a, Immuno uorescence of perk in Tg(mpx:mCherry) at 30 minutes post wounding. b, Immuno uorescence of perk in Tg(mpx:mCherry) with/without lyn morpholino at 30 minutes post wounding. c, Quanti cation of uorescence intensity of perk in neutrophils (ctrl: 45 cells (4 larvae), lyn MO: 21 cells (4 larvae)). d, Immuno uorescence of perk and Mpx in Tg(mpx:lyn WT-GFP) with/without lyn MO at 30 minutes post wounding. e, Immuno uorescence of 16 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH perk and Mpx in Tg(mpx:lyn C466A-GFP) with/without lyn MO at 30 minutes post wounding. f, Quanti cation of uorescence intensity of perk in neutrophils in Tg(mpx:lyn WT-GFP) (Lyn-GFP(+)/Ctrl: 95 cells (11 larvae), Lyn-GFP(+)/lyn MO: 99 cells (11 larvae)). g, Quanti cation of uorescence intensity of perk in neutrophils in Tg(mpx:lyn C466A-GFP) (C466A-GFP(+)/Ctrl: 128 cells (25 larvae), C466A-GFP(+)/lyn MO: 64 cells (23 larvae)). *P<0.05, two-tailed unpaired t-test (c,f).scale bars: 10 μm. www.nature.com/nature 17
RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 12 Erk inhibitors impair neutrophil accumulation at wounds. a, 3 dpf larvae were treated with DMSO, 100 μm PD98059 or 20 μm U0126, wounded, xed 30 minutes post wounding and stained with Sudan Black. Erk inhibitors impair neutrophil accumulation at wounds (DMSO: 23 larvae, PD98059: 22 larvae, U0126: 23 larvae). b, Representative pictures of Sudan Black staining. *P<0.05, one-way ANOVA with Dunnett post-test 18 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 13 DNA sequences of codon-optimized zebra sh lyn and human LYN. a, DNA sequence of codon-optimized zebra sh lyn. b, DNA sequence of www.nature.com/nature 19
RESEARCH SUPPLEMENTARY INFORMATION codon-optimized human LYN. Codon optimization was performed by GenScript (Piscataway, NY). 20 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH Supplementary movie 1 Movie shows time-lapse imaging of H 2 O 2 immediately after tail transection in 2.5 dpf larvae expressing HyPer probe. The larvae were treated with DMSO (left) or 20 μm PP2 (right) for 1h before imaging and images were taken with the drug in E3. Supplementary movie 2 Movie shows time-lapse imaging of neutrophil random migration in the cephalic mesenchyme of 3 dpf Tg(mpx:Dendra2). The larvae were treated with DMSO (left) or 20 μm PP2 (right) for 1h before imaging and images were taken with the drug in E3. Supplementary movie 3 Movie shows four examples of representative time-lapse imaging of 2.5 dpf Tg(mpx:Dendra2) injected with lyn MO or buffer. Note increased total numbers of neutrophils in the lyn morphants. Neutrophils migrate to wounds immediately after wounding in control but not in lyn morphants. Supplementary movie 4 Movie shows LTB 4 -mediated neutrophil dissemination into the ns of zebra sh larvae. In the rst movie, 3 dpf larvae were pretreated with DMSO or DPI, and bathed in E3 containing 30 μm LTB 4. In the second movie, 2.5 dpf larvae injected with lyn MO or buffer were bathed in E3 containing 30 μm LTB 4. 1. Niethammer, P., Grabher, C., Look, A.T. & Mitchison, T.J. A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebra sh. Nature 459, 996-999 (2009). 2. Scapini, P., Pereira, S., Zhang, H. & Lowell, C.A. Multiple roles of Lyn kinase in myeloid cell signaling and function. Immunol Rev 228, 23-40 (2009). www.nature.com/nature 21
RESEARCH SUPPLEMENTARY INFORMATION 3. Yoo, S.K. & Huttenlocher, A. Spatiotemporal photolabeling of neutrophil traf cking during in ammation in live zebra sh. J Leukoc Biol 89, 661-667. 22 WWW.NATURE.COM/NATURE