SUPPLEMENTARY INFORMATION

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1 In the format provided by the authors and unedited. SUPPLEMENTARY INFORMATION ARTICLE NUMBER: DOI: /NMICROBIOL Dual-specificity phosphatase 6 deficiency regulates gut microbiome and transcriptome response against diet-induced obesity in mice Jhen-Wei Ruan, Sarah Statt, Chih-Ting Huang, Yi-Ting Tsai, Cheng-Chin Kuo, Hong-Lin Chan, Yu-Chieh Liao, Tse-Hua Tan, Cheng-Yuan Kao NATURE MICROBIOLOGY Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Supplementary Figures Supplementary Figure 1. Dusp6-deficiency increased glucose tolerance and alleviated obesity-associated phenotypes in mice.

2 Supplementary Figures Supplementary Figure 1. Dusp6-deficiency increased glucose tolerance and alleviated obesity-associated phenotypes in mice. a, Glucose tolerance test after 12 hours fasting of male WT/dusp6 KO mice were fed on CD or HFD for 16 weeks. Data are presented as the mean ± SEM of each time point. WT-CD, N=17; WT-HFD, N=15; dusp6 KO-CD, N=17; dusp6 KO-HFD, N=13. b, Insulin tolerance test after 6 hours fasting of male WT/dusp6-KO mice were fed on CD or HFD for 16 weeks. Data are presented as the mean ± SEM of each time point. WT-CD, N=7; WT-HFD, N=7; dusp6 KO-CD, N=5; dusp6 KO-HFD, N=5. c, Blood glucose level after 6 hours fasting of male WT/dusp6-KO mice fed on CD or HFD for 16 weeks. Data are presented as the mean ± SEM. WT-CD, N=7; WT-HFD, N=7; dusp6 KO-CD, N=7; dusp6 KO-HFD, N=6. d, Weight of flank adipose tissue of male WT/dusp6-KO mice fed on CD or HFD for 16 weeks. Data are presented as the mean ± SEM. WT-CD, N=10; WT-HFD, N=8; dusp6 KO-CD, N=9; dusp6 KO-HFD, N=7. For (c) and (d), asterisks indicate statistical difference (*P<0.05, ***P<0.0005) according to One-Way ANOVA analysis and Tukey post-hoc test. e-f, Haematoxylin & eosin (H&E)-stained sections of flank adipose tissue (e) and liver tissue (f) from male WT and dusp6-ko mice fed on CD or high-fat diet HFD for 16 weeks. Scale bar, 100 µm. 2

3 Supplementary Figure 2. Fecal/gut microbiome composition of FMT recipient mice. a, Schematic diagram of fecal microbiome transplantation, metabolic tests and tissue sampling of WT/D6KO microbiota recipient mice. b, Quantitative PCR validation of 16S rrna gene of all eubacteria before and after 16 weeks of HFD treatment. The plasmid contained 16S rrna sequence of Eubacteria was used as the standard to validate the absolute copy number of each sample. The number of mice for each group: before HFD treatment, WT, N=18 and dusp6ko, N=17; after 16 weeks of HFD treatment, WT, N=8 and dusp6ko, N=7. The red line indicates the mean of each group. Double asterisks and triple asterisks indicate statistical difference (P<0.005 and P<0.0005) of mean± SEM according to Mann-Whitney analysis. c-d, Weighted-UniFrac (c) and Unweighted-UniFrac (d) Principle coordinates analysis (PCoA) plot represents changes between chow diet (T0) and HFD (T16) fed wild-type and dusp6 knockout mice. The number of mice per group: T0: WT-CD, N=11; dusp6 KO-CD, N=13; T16: WT-HFD, N=6; dusp6 KO-HFD, N=6. e, Composition of Firmicutes family of WT/dusp6 KO microbiota recipient mice before and after 16 weeks of HFD treatment. f, Composition of Proteobacteria 3

4 family of WT/dusp6 KO microbiota recipient mice before and after 16 weeks of HFD treatment. g, Cladogram generated from LEfSe analysis (α < 0.1 for factorial Kruskal-Wallis test), showing the most differentially abundant microbial clades enriched in microbiota from wild-type (green) or dusp6 knockout (red) FMT recipient mice fed HFD (T16). WT-HFD, N=8; dusp6 KO-HFD, N=8. h, LDA scores of the differentially abundant microbial clades (with LDA score >2 and significance of α < 0.1 determined by Kruskal-Wallis test) shown in (g). i, 5-6 weeks old male germ-free mice were oral gavaged with BHI broth culture of fecal microbiota derived from representative donors and then fed on HFD for 16 weeks. Body weights were monitored weekly. Data are presented as the mean ± SEM of each time point. CFMT-WT, N=11; CFMT-dusp6 KO, N=12. j, Weight gain of WT/dusp6 KO CFMT recipient mice after 16 weeks of HFD treatment. Data are presented as the mean ± SEM. The asterisk indicates statistical difference (P<0.05) according to Mann-Whitney analysis. CFMT-WT, N=11; CFMT-dusp6 KO, N=12. 4

5 Supplementary Figure 3. Transcriptome profile of CD or HFD treated WT/dusp6-KO mice. a-b, Immuno-histochemistry analysis (a) and qrt-pcr analysis (b) of DUSP6 protein or dusp6 gene expression in intestine of chow diet of HFD fed dusp6 KO mice. Scale bar, 100 µm. c-d, The number of overall DEGs (c) and gene ontology biological processes (d) that were significantly enriched among up-regulated genes or down-regulated genes in the comparisons of HFD-fed WT and dusp6-ko mice. e, The non-sense mutations of both alleles of DUSP6 gene of DUSP6 KO cells were confirmed by DNA sequencing analysis. (Green letters are not included in grna but recognized by Cas9 protein). f, Immunoblot analysis of DUSP6 protein in WT and DUSP6 KO Caco-2 cells. g, TEER analysis of WT and DUSP6 KO Caco-2 cells after 21 days of differentiation. Data are presented as the mean ± SEM (N=3 separate experiments). The asterisk indicates statistical difference (P<0.05) according to two-tailed t-test. h, 2 hours after oral gavaged with FITC conjugated 4kDa dextran, mouse serum was collected for fluorescence measurement. Data are presented as the mean ± SEM. N=11 for each group. The asterisk indicates statistical difference (P<0.05) according to Mann-Whitney analysis. i-j, 5

6 qrt-pcr validations of the KEGG Ppar pathway (i) and tight junction pathway (j) that were significantly enriched in the up-regulated genes of the comparison of dusp6-deficient mice to wild-type mice in control diet in CD fed FMT recipient mice. Data are presented as the mean ± SEM. N=8 for FMT-WT recipient mice and N=7 for FMT-Dusp6 KO recipient mice. The asterisk indicates statistical difference (P<0.05) according to Mann-Whitney analysis. ns indicates statistically non-significant. 6

7 Supplementary Figure 4. KEGG immunological pathways down-regulated in HFD-fed dusp6-deficient mice. a, DAVID KEGG pathway analysis of down-regulated DEGs in WT-HFD/D6KO-HFD dataset. b, The down-regulated DEGs in HFD-fed dusp6-deficient mice involved in KEGG cytokine-cytokine receptor interaction pathway. c, qrt-pcr analysis of Ltα, Ltβ and LTβR of WT and dusp6-deficient mice before and after HFD treatment. For (c), data are presented as the mean ± SEM. Asterisk (P<0.05) and double asterisks (P<0.005) indicate statistical difference according to One-Way ANOVA analysis and Tukey post-hoc test. The number of mice for each group: WT-CD, N=7; WT-HFD, N=8; Dusp6KO-CD, N=7; Dusp6KO-HFD, N=8. 7

8 Supplementary Figure 5. Dusp6-deficiency reversed HFD-mediated induction of intestinal AMPs. a, qrt-pcr analysis of intestinal TJP-1 gene of WT/dusp6-deficient mice before and after HFD treatment. Data are presented as the mean ± SEM. ns indicates statistical non-significant according to One-Way ANOVA analysis and Tukey post-hoc test. N=5 for each group. b, Serum endotoxin levels of WT/dusp6-deficient mice before and after HFD treatment were determined by LAL test. Data are presented as mean ± SEM. Double asterisks indicate statistical difference (P<0.005) according to One-Way ANOVA analysis and Tukey post-hoc test. The number of mice for each group: WT-CD, N=9; WT-HFD, N=13; Dusp6KO-CD, N=9; Dusp6KO-HFD, N=13. c-d, Quantitative RT-PCR validation of RegIIIβ (c) and RegIIIγ (d). Data are presented as the mean ± SEM. The number of mice for each group: WT-CD, N=10; WT-HFD, N=8; Dusp6KO-CD, N=9; Dusp6KO-HFD, N=7. ns indicates statistically non-significant according to One-Way ANOVA analysis and Tukey post-hoc test. 8

9 Supplementary Tables Supplementary Table 1. Taxonomic composition at phylum and family level of fecal/gut microbiota of WT and dusp6 KO mice before and after HFD treatment. Dusp6 WT-Chow Dusp6 WT-HFD KO-Chow KO-HFD Samples Good s coverage (phylum) Shannon index (phylum) 98.22% 98.5% 98.48% 98.83% PERMANOVA /Adonis test Phylum (%) Chow diet fed (WT vs. Dusp6 KO) R2= , p=0.002 HFD fed (WT vs. Dusp6 KO) R2= , p=0.003 Actinobacteria 0.03± ±0.02* 0.24± ±0.05 Bacteroidetes 42.13± ±2.81* 62.94± ±5.27* Cyanobacteria 0.08± ± ± ±0.03* Deferribacteres 3.62± ±0.24** 1.05± ±1.32 Firmicutes 53.2± ±2.79* 34.62± ±2.11* Proteobacteria 0.59± ±0.10* 1.01± ±2.30 TM7 0.02± ± ± ±0.00 Tenericutes 0.35± ± ± ±0.01 Family (%) Bacteroidaceae 2.24± ± ± ±1.63** S ± ±3.26** 21.90± ±2.51 Rikenellaceae 9.81± ± ± ±0.95 Porphyromonadaceae 0.07± ± ± ±0.79 Odoribacteraceae 1.99± ± ± ±1.46 Ruminococcaceae 10.30± ±0.90* 11.65± ±1.62 9

10 Erysipelotrichaceae 0.24± ± ± ±1.70** Lachnospiraceae 12.43± ±0.97** 3.23± ±0.39 Lactobacillaceae 2.72± ± ± ±0.34 Clostridiaceae 1.22± ± ± ±0.02 Data are presented as the mean ± SEM. Asterisk (P<0.05) and double asterisks (P<0.005) indicate statistical difference according to Mann-Whitney analysis performed on the comparison of Chow diet fed WT vs. Dusp6 KO mice or HFD fed WT vs. Dusp6 KO mice data set. 10

11 Supplementary Table 2. Taxonomic composition at phylum and family level of fecal/gut microbiota of WT and D6KO microbiota recipient mice before and after HFD treatment. Dusp6 WT-Chow Dusp6 WT-HFD KO-Chow KO-HFD Samples Good s coverage (phylum) Shannon index (phylum) 99.15% 99.2% 99.06% 99.13% PERMANOVA /Adonis test Phylum (%) Chow diet fed FMT (WT vs. D6KO) R2= , p=0.005 HFD fed FMT (WT vs. D6KO) R2= , p=0.065 Actinobacteria 0.49± ± ± ±0.07 Bacteroidetes 45.12± ± ± ±2.90 Cyanobacteria N.D. N.D. 0.05± ±0.01 Deferribacteres 0.86± ± ± ±0.42 Firmicutes 50.37± ± ± ±2.74 Proteobacteria 3.13± ± ± ±0.08 TM7 0.01± ±0.00** 0.01± ±0.00 Tenericutes 0.02± ± ± ±0.01 Family (%) Bacteroidaceae 22.74± ± ± ±1.62 S ± ± ± ±0.89 Rikenellaceae 1.44± ± ± ±0.59 Porphyromonadaceae 4.48± ± ± ±0.17 Odoribacteraceae 0.17± ± ± ±0.51 Ruminococcaceae 6.69± ± ± ±2.00 Erysipelotrichaceae 21.03± ± ± ±4.37 Lachnospiraceae 3.58± ± ± ±

12 Lactobacillaceae 13.09± ± ± ±2.02 Clostridiaceae 0.07± ± ± ±0.00 Dehalobacteriaceae 0.21± ± ± ±0.03 Enterococcaceae 0.04± ± ± ±0.16 Turicibacteraceae 0.00± ±0.03** 0.08± ±0.05 Peptococcaceae 0.02± ± ± ±0.04 Streptococcaceae 0.05± ± ± ±0.05** Desulfovibrionaceae 0.21± ± ± ±0.05 Helicobacteraceae 0.06± ± ± ±0.05 Alcaligenaceae 2.63± ± ± ±0.00 Enterobacteriaceae 0.22± ± ±0.00* Data are presented as the mean ± SEM. Double asterisks (P<0.005) indicate statistical difference according to Mann-Whitney analysis performed on the comparison of Chow diet fed WT vs. D6KO FMT recipient mice or HFD fed WT vs. D6KO FMT recipient mice data set. N.D.: Non-Detectable. 12

13 See the supplemented excel file for Supplementary Table 3. Supplementary Table 4. qrt-pcr primers for mouse genes and qpcr primers for bacteria Gene name Direction Sequence Reference TBP1 Pparg Adipoq Pck1 CD36 Cyp27a1 Scd1 Me1 Fabp4 Angptl4 Cldn1 Jam2 Jam3 Ppm1j Pard6g Myh10 5'-caaacccagaattgttctcctt-3' 5'-atgtggtcttcctgaatccct-3' 5'-gtgccagtttcgatccgtaga-3' 5'-ggccagcatcgtgtagatga-3' 5'-ccatctggaggtgggagac-3' 5'-ctgcatagagtccattgtggtc-3' 5'-tgctgcccttatcattaggttt-3' 5'-gggtgcagaatctcgagttg-3' 5'-ggagcaactggtggatggtt-3' 5'-ttgagactctgaaaggatcagca-3' 5'-ccacaagggcctcacctatg-3' 5'-gcacctggtccagccgggtg-3' 5'-ccgggagaatatcctggttt-3' 5'-cactggcagagtagtcgaagg-3' 5'-ctcataggagttgctgcaattgg-3' 5'-cgttgaaggcagccatatcc-3' 5'-aaggtgaagagcatcataaccct-3' 5'-tcacgcctttcataacacattcc-3' 5'-acggccaatgagctggg-3' 5'-gggcagggaaaggcca-3' 5'-actccttgctgaatctgaacagt-3' 5'-ggacacaaagattgcgatcag-3' 5'-tgtttgtactacatgcgaacctg-3' 5'-tccatttccagggagtctga-3' 5'-ggctgcgacttcgactgta-3' 5'-gagattcactgcctctatcatgc-3' 5'-tctctcgacccacctttctg-3' 5'-ttgccagcattgatgacct-3' 5'-tcggctatgctgatgtgc-3' 5'-gctgtaatggtctgcttcttctc-3' 5'-tgctggttttgaaatttttgag-3' 5'-tgccctctcgctggtact-3'

14 14 Gnai1 5'-acgattcggcagcgtactat-3' 5'-atcctgctgagttgggatgt-3' Rras 5'-tcacaagctggtggtcgtag-3' 5'-tgggatcatagtcagacacaaag-3' Mras 5'-ctctcctgtgccgagtgg-3' 5'-actcctcagggctttggtct-3' Lta 5'-cacactgccgcttcctctat-3' 5 5'-gccgagcagtgtcatgtg-3' Ltb 5'-cctggtgaccctgttgttg-3' 5 5'-tgctcctgagccaatgatct-3' LtbR 5'-ccataccagatgtgagatccag-3' 5'-atggccagcagtagcattg-3' TJP1 5'-atgcagacccagcaaagg-3' 5'-tggttttgtctcatcatttcttca-3' Defa5 5'-agctggctgactgggtgtgt-3' 6 5'-cggtgcttcggtctccacgg-3' RegIIIb 5'-atggctcctactgctatgcc-3' 5'-gtgtcctccaggcctcttt-3' RegIIIg 5'-atggctcctattgctatgcc-3' 7 5'-gatgtcctgagggcctctt-3' Eubacteria UniF340 5'- actcctacgggaggcagcagt-3' 8 UniR514 5'- attaccgcggctgctggc-3' Akkermansia muciniphila 5'-cagcacgtgaaggtggggac-3' 9 5'-ccttgcggttggcttcagat-3' SFB SFB736F 5'-gacgctgaggcatgagagcat-3' 10 SFB844R 5'-gacggcacggattgttattca-3'

15 Supplementary References 1. Wang, F., Wang, J., Liu, D. & Su, Y. Normalizing genes for real-time polymerase chain reaction in epithelial and nonepithelial cells of mouse small intestine. Analytical biochemistry 399, (2010). 2. Galmozzi, A. et al. ThermoMouse: an in vivo model to identify modulators of UCP1 expression in brown adipose tissue. Cell reports 9, (2014). 3. Jais, A. et al. Heme oxygenase-1 drives metaflammation and insulin resistance in mouse and man. Cell 158, (2014). 4. Vergnes, L., Lee, J.M., Chin, R.G., Auwerx, J. & Reue, K. Diet1 functions in the FGF15/19 enterohepatic signaling axis to modulate bile acid and lipid levels. Cell metabolism 17, (2013). 5. Zotes, T.M. et al. PI3K p110gamma deletion attenuates murine atherosclerosis by reducing macrophage proliferation but not polarization or apoptosis in lesions. PloS one 8, e72674 (2013). 6. Sommer, F. & Backhed, F. The gut microbiota engages different signaling pathways to induce Duox2 expression in the ileum and colon epithelium. Mucosal immunology 8, (2015). 7. Shiu, J. et al. Gastric LTi cells promote lymphoid follicle formation but are limited by IRAK-M and do not alter microbial growth. Mucosal immunology 8, (2015). 8. Barman, M. et al. Enteric salmonellosis disrupts the microbial ecology of the murine gastrointestinal tract. Infect Immun 76, (2008). 9. Everard, A. et al. Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity. Proc Natl Acad Sci U S A 110, (2013). 10. Snel, J. et al. Comparison of 16S rrna sequences of segmented filamentous bacteria isolated from mice, rats, and chickens and proposal of "Candidatus Arthromitus". Int. J. Syst. Bacteriol. 45, (1995). 15

Supplementary Figures and Tables

Supplementary Figures and Tables Supplementary Figure 1. Intestinal epithelial MyD88 deletion decreases fat mass under HFD. (a) Final fat mass expressed as a percentage of final body weight (n=25). (b)