Babaev et al. SUPPLEMENTAL DATA. Oil-Red-O MOMA-2. Lesion Area (μm 2 x10 3 ) Lesion Area(% of total) WT Akt1 -/- WT Akt1 -/-

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1 SUPPLEMENTL T Oil-Red-O MOM-2 WT Ldlr -/- kt1 -/- Ldlr -/- WT Ldlr -/- kt1 -/- Ldlr -/- Lesion rea(% of total) Lesion rea (μm 2 x1 3 ) WT kt1 -/- WT kt1 -/- Figure S1. Hematopoietic kt1 deficiency has no impact on early atherosclerosis in male Ldlr -/- mice. (,) therosclerotic lesions in the distal () and proximal () aorta of mice reconstituted with WT (n=9) or kt1 -/- (n=11) FL. ortas were pinned out en face and stained with Sudan IV (); serial sections were stained with Oil-Red-O or MOM-2. Scale bars, 2 μm; a pin size, 1 mm. (,) The extent of atherosclerotic lesions in the distal and proximal aortas of Ldlr -/- mice reconstituted with WT( ) or kt1 -/- ( ) FL.

2 SUPPLEMENTL T E F Ly-6G PI Merge G 14 Ly-6G+ cells numbers Lesions dventitia Figure S2. Fluorescent staining of neutrophils in atherosclerotic lesions of Ldlr -/- mice transplanted with WT (-) or kt2 -/- (-F) FL. (-F) 5-micron sections of the aortic sinus were fixed with acetone and stained with the rat anti-mouse antibody to Ly-6G (18 clone; iolegend) followed by lexa Flour 594 goat anti-rat antibodies. Scale bars, 1 μm. (G) Percent of Ly-6G-positive cells in atherosclerotic lesions and aortic adventitia of WT Ldlr -/- ( ) and kt2 -/- Ldlr -/- ( ) mice after 8 weeks of the Western diet.

3 SUPPLEMENTL T WT kt1 -/- kt2 -/- 5+ cells numbers Lesions dventitia Figure S3. 5-positive cells in atherosclerotic lesions and aortic adventitia of Ldlr -/- mice reconstituted with WT, kt1 -/- and kt2 -/- FL. (-) 5-micron sections of the proximal aorta were fixed with acetone and stained with the rat anti-mouse bs to 5 (Ly1.2; PharMingen) followed by avidin-biotin complex labeled with alkaline phosphatase. Scale bars, 5 μm.() Percent of 5-positive cells in atherosclerotic lesions and adventitia of WT Ldlr -/- ( ), kt1 -/- Ldlr /- ( ) and kt2 -/- Ldlr -/- ( ) mice fed with the Western diet for 16 weeks..

4 SUPPLEMENTL T 45R/22 P- 45R/22 P- 11b (percent) 45R/22 P PE- WT d11b+/ly6g- 45R/22 (%) E PE- kt2 -/- kt1-/- 45R/22+ F 9.2 (percent) PE Figure S4. Flow cytometry analysis of blood monocyes, - and T-cells in Ldlr -/- mice transplanted with WT, kt1 -/- and kt2 -/- FL. (-) nalysis of the blood cell surface markers 45R/22 (blue color) and 9.2 (violet color) in WT (), kt1 -/- () and kt2 -/- () mice. lood was isolated by retro-orbital bleeding, erythrocytes were hemolyzed by the lysing buffer and cells were analyzed by multicolor flow cytometry using bs to 11b FIT, 45R/22 P and 9.2 PE. (-F) Percent of 11b+/Ly6G- monocytes (), 45R/22+ (E) and 9.2+ (F) blood cells in recipient mice reconstituted with WT ( ), kt1 -/- ( ) and kt2 -/- ( ) FL.

5 SUPPLEMENTL T R2 P- R2 P- R2 P- inos FIT- WT inos FIT- kt1 -/- inos FIT- kt2 -/- F 115+ (percent) inos+ (percent) R2+/iNOS+(%) E inos R2+/iNOS+ Figure S5. 115, inos and R2 expression in monocytes isolated from Ldlr -/- mice reconstituted with WT, kt1 -/- and kt2 -/- FL on a chow diet. (-) 115- gaited flow cytometry analysis of R2 and inos expression in blood monocytes isolated from mice with WT (), kt1 -/- () and kt2 -/- () FL. (-F) Percent of 115+, inos+ and inos+/r2+ monocytes in Ldlr -/- mice reconstituted with WT( ), kt1 -/- ( ) and kt2 - /- ( ) FL (p<.5 by One Way nalysis of Variance, Tukey test).

6 SUPPLEMENTL T R2 P- R2 P- R2 P- Ly-6 FIT- WT Ly-6 FIT- kt1 -/- Ly-6 FIT- kt2 -/- 11b+ (percent) Ly-6 hi (percent) E Ly-6 lo (percent) F b+ Ly-6 hi Ly-6 lo Figure S6. 11b, Ly-6 hi and Ly-6 lo expression in monocytes isolated from Ldlr -/- mice reconstituted with WT, kt1 -/- and kt2 -/- FL after 6 weeks of Western diet. (-) 11b-gaited flow cytometry analysis of Ly-6 in blood monocytes isolated from mice with WT (), kt1 -/- () and kt2 -/- () FL. (-F) Percent of 11b+, Ly-6 hi and Ly- 6 lo monocytes from Ldlr -/- mice reconstituted with WT( ), kt1 -/- ( ) and kt2 -/- ( ) FL (p<.5 by One Way nalysis of Variance, Tukey test).

7 SUPPLEMENTL T WT kt1 -/- kt2 -/- p-kt S 473 β-actin kt1 kt2 pan-kt Ins p-kts 473 /β-actin ratio Ins WT kt1 -/- kt2 -/- Figure S7. kt signaling in WT( ), kt1 -/- ( ) and kt2 -/- ( ) cells in response to insulin. () WT, kt1 -/- and kt2 -/- peritoneal macrophages were untreated or treated with insulin (1nM) for 1 or 15 min. Proteins were extracted, resolved and analyzed by Western blot. () The ratio of p-kt/b-actin is presented as the average (mean+sem) of three different experiments (p <.5 compared to untreated WT cells by One Way nalysis of Variance).

8 SUPPLEMENTL T Ratio rg1/β-actin IL Ratio rg1/β-actin IFNγ Ratio Ym1/β-actin IL Figure S8. Priming WT, kt1 -/- and kt2 -/- macrophages to M1 or M2 phenotype. WT ( ), kt1 -/- ( ) and kt2 -/- ( ) peritoneal macrophages were untreated () or treated with IL-4 (,) or IFNγ () for 24 and 48 hours; Graphs represent data of the Western blot analysis (Fig 4); p<.5 compared to control WT cells at the same time point by One Way nalysis of Variance, Tukey test.

9 Table S1. LPS-altered and kt2-dependent mirns in peritoneal macrophages from WT mice mirn Fold hange P-Value mmu-mir mmu-mir mmu-mir hsa-mir-151-5p rno-mir-196c mmu-mir-18a-3p mmu-mir-674-3p mmu-mir-362-5p hsa-mir-125a-5p hsa-mir-126-5p hsa-mir hsa-mir-146b hsa-mir-338-3p hsa-mir mmu-mir p mmu-mir mmu-mir-31-3p