Figure S1- Targeting strategy for generating SIK1-T182A, SIK2-T175A and SIK3-T163A KI mice.

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1 Figure S1- Targeting strategy for generating SIK1-T12A, SIK2-T175A and SIK3-T163A mice. Left panel represents a depiction of the genomic loci of each gene, the targeting strategy and the final recombined product. Right panel shows the results of Southern blotting demonstrating successful targeting of the genomic locus. (A) SIK1-T12A, (B) SIK2-T175A and () SIK3-T163A. Figure S2- SIK3 mice were present at sub-mendelian ratios post-weaning and are smaller than their littermates. (A) SIK3 embryos are present at Mendelian ratios at E15.5. Matings were set up between SIK3 heterozygous mice; embryos were harvested from pregnant females at E15.5 and genotyped. There was no significant difference (p>.5, n=5) between observed and expected ratios of SIK3 embryos as determined by hi-square test. (B) SIK3 mice are present at sub-mendelian ratios post-weaning. Matings were set up between SIK3 heterozygous mice and genotypes of the progeny were determined post-weaning (day 21-2). There was a significant difference (p<.1, n=5) between the observed and expected ratios of the genotypes as determined by hi-square test. (,D) SIK3 mice are smaller than their littermates. Mice were weighed at weaning (left panel) and weights plotted according to gender and genotype (mean ± SD, n=16 male or n=22 female). Mice were weighed every 7 days post-weaning (right panel), and weights were plotted according to gender and genotype (mean ± SD, n=-). Statistics represent analysis by two-way ANOVA and Bonferroni post-tests comparing SIK3 mice to. (HET, heterozygous) Figure S3- SIK inhibition elevates IL-6 production in macrophages at early time points. BMDMs were treated for 1 h with vehicle control or 5 nm HG1-1 prior to stimulation with ng/ml LPS for the indicated times. (A, ) IL-6 mrna levels were determined by qpr and expressed as fold-change relative to untreated control cells (mean ± SEM, n=). (B, D) IL-6 concentration was determined in cell-free culture supernatants using the Bio-Plex Pro Assay System (mean ± SEM, n=). Statistics represent analysis by two-way ANOVA followed by Bonferroni post-tests. Figure S- SIK2/3 double embryos are present at Mendelian ratios at E15.5. Matings were set up between SIK2 ki/ki SIK3 wt/ki mice; embryos were harvested from pregnant females at E15.5 and genotyped. There was no significant difference (p>.5, n=67) between observed and expected ratios of embryos as determined by hi-square test. Figure S5- ytokine production in SIK1/2 double macrophages stimulated with Pam 3 SK. BMDMs from, SIK1, SIK2 and SIK1/2 double mice were stimulated with 1 µg/ml Pam 3 SK for (A) h (IL-) or (B-D) h (TNFα, IL-6 and IL-12p). The concentrations of cytokines in cell-free culture supernatants were measured using the Bio-Plex Pro Assay System (mean ± SEM, n=). Statistics represent analysis by one-way ANOVA, followed by Bonferroni posttests. Figure S6- Macrophages differentiated in the presence of a SIK inhibitor express regulatory phenotype markers. (A-) Bone marrow cells were differentiated in the presence of vehicle control (blue/red bars) or nm HG1-1 (green/purple bars) in media containing M-SF. On day 7, macrophages were harvested and either re-plated in the presence of vehicle control (blue/red/green bars) or retained in the drug (purple bars). h later, macrophages were treated with vehicle control (blue/green/purple bars) or nm HG1-1 (red bars) for 1 h. All cell populations were then stimulated with ng/ml LPS for 2- h. The relative levels of Arg1 ( h) (A), LIGHT (2 h) (B) and SPHK1 (2 h) () mrna were determined by qpr. qpr results are expressed as fold-induction relative to control cells that were not treated with drug or LPS. Results were generated using cells pooled from mice. Error bars represent the SEM for 3 independent stimulations. Data are representative of three independent experiments. Statistics represent analysis by unpaired student s t-test (A) or two-way ANOVA followed by Bonferroni post-tests (B, ). Figure S7- SIK inhibition has little effect on NO production in macrophages. (A) Kinetics of production in LPS-stimulated BMDMs from SIK1 or SIK2 mice compared to. (B) Kinetics of production in LPS-stimulated FLDMs from SIK3 mice compared to

2 . (A,B) Macrophages were stimulated with ng/ml LPS for the indicated times; mrna levels were determined by qpr and expressed as fold-change relative to unstimulated cells (mean ± SEM, n=). () FLDMs from and SIK3 mice were stimulated with ng/ml LPS for h and cell extracts were immunoblotted with the indicated antibodies. (D) Nitrite and nitrate levels in cell-free culture supernatants from and SIK3 FLDMs stimulated with ng/ml LPS for h were determined as described in Materials and Methods (mean ± SEM, n=3). (E-G) FLDMs from mice were treated with 5 nm HG1-1 for 1 h prior to stimulation with ng/ml LPS for the indicated times. Experiments were performed as described above. (E) content (mean ± SEM, n=). (F) inos protein content. (G) Nitrite and nitrate levels following h LPS stimulation (mean ± SEM, n=3). (H) NO production in BMDMs treated as in G (mean ± SEM, n=3). (I) FLDMs were treated with 5 nm HG1-1 or vehicle control for 1 h prior to stimulation with ng/ml LPS and ng/ml IFNγ for h. Nitrite and nitrate levels were measured as in D (mean ± SEM, n=3). Statistics represent analysis by two-way ANOVA followed by Bonferroni post-tests. Figure S- SIK1, SIK2 and SIK3 kinase activity in mouse embryos. embryos were harvested from pregnant females at E16.5 and homogenized in lysis buffer. The catalytic activity of each SIK isoform was measured as described in Materials and Methods (mean ± SEM, n=3).

3 Supplementary Figure S1 A 5 probe Eco R1 digest 3 probe Pac I / Mfe I digest.2 kb 5.5 kb 13.6 kb 9.3 kb B 5 probe Bam HI digest 3 probe Eam 11O5I digest.2 kb 25 kb.1 kb 13.3 kb 5 probe Xma I digest 3 probe Xma I digest 1 kb. kb 1 kb 7 kb

4 Supplementary Figure S2 A Genotype Numbers % Observed % Expected SIK3 wt/wt SIK3 wt/ki SIK3 ki/ki hi-square test p>.5, not significant B Genotype Numbers % Observed % Expected SIK3 wt/wt SIK3 wt/ki SIK3 ki/ki hi-square test p<.1 Weight (g) 15 5 Male Weight (g) ** HET D Weight (g) 15 5 Female Weight (g) HET Het Genotype Week Het Genotype Week

5 Supplementary Figure S3 A IL-6 mrna 15 5 ** B IL6 (pg/ml) 5 ontrol HG1-1 IL-6 mrna LPS (min) D IL6 (pg/ml) LPS (min) 15 5 ontrol HG

6 Supplementary Figure S Genotype Numbers % Observed % Expected SIK2 ki/ki SIK3 wt/wt SIK2 ki/ki SIK3 wt/ki SIK2 ki/ki SIK3 ki/ki hi-square test p>.5, not significant

7 Supplementary Figure S5 A B 25 IL- (pg/ml) TNFα (pg/ml) 15 5 IL-6 (pg/ml) 5 SIK1 ki/ki SIK1 ki/ki SIK2 ki/ki SIK1 ki/ki /SIK2 ki/ki SIK2 ki/ki SIK1 ki/ki /SIK2 ki/ki D IL-12p (pg/ml) 15 5 SIK1 ki/ki SIK1 ki/ki SIK2 ki/ki SIK1 ki/ki /SIK2 ki/ki SIK2 ki/ki ** SIK1 ki/ki /SIK2 ki/ki

8 Supplementary Figure S6 A B LIGHT Arg1 mrna * LIGHT mrna * SPHK1 mrna Vehicle control Acute HG1-1 HG1-1 differentiation hronic HG1-1 Vehicle control Acute HG1-1 HG1-1 differentiation hronic HG1-1 Vehicle control Acute HG1-1 HG1-1 differentiation hronic HG1-1

9 Supplementary Figure S7 SIK1ki/ki Nitrite Nitrate H I n.s Nitrate Nitrite Nitrate 6 Nitrate Nitrite ki/ki ontrol HG1-1 5 SIK3ki/ki G SIK3 12 E Nitrate 5 Nitrite 6 Nitrite ol H G 1 6 tr Nitrate 15 Nitrite on 16 GAPDH 12 HG1-1 inos F ol H G 1 GAPDH LPS inos D SIK3ki/ki tr 6 on ontrol HG1-1 ol H G 1 tr 6 on SIK3ki/ki 1 B SIK2ki/ki A SIK3ki/ki SIK3ki/ki 12 16

10 Supplementary Figure S munits/mg lysate 6 2 SIK1 Embryo SIK2 SIK3