Reltive expression 5 1 15 2-5 5 (n = 3) (n = 3) GFM nd PFM PFM GFM mmu-mir-145 mmu-mir-143 mmu-mir-72 mmu-mir-22 mmu-mir-27 mmu-mir-125-5p mmu-mir-23 mmu-mir-29 mmu-mir-126-3p mmu-let-7d mmu-let-7c mmu-mir-199-3p mmu-mir-29 mmu-mir-148 mmu-let-7 mmu-let-7g mmu-let-7 mmu-mir-29c mmu-mir-14 mmu-mir-16 mmu-mir-451 mmu-mir-23 mmu-mir-125-5p mmu-mir-99 mmu-mir-199-5p mmu-mir-21 mmu-mir-15 mmu-let-7f mmu-mir-365 mmu-mir-3 mmu-mir-181 mmu-mir-1 mmu-mir-13 mmu-mir-26 mmu-mir-152 mmu-let-7e mmu-let-7i mmu-mir-3d mmu-mir-1 <2 mirs represent > 8 % of mirs with <.6 % vrition etween c % of totl glol mir expression % Chnge (PFM vs. GFM) d 1.5 1 PFM GFM (n = 3) ns ns.5 Up-regultion Supplementry Fig. 1. Aortic micrornas re differentilly expressed in PFM v. GFM. () Het mp of differentilly regulted micrornas in orts of PFM nd GFM. Ech column represents one mouse. n = 3 in ech group. () Percent expression of most undnt micrornas in orts of PFM. n = 3 for ech microrna. (c) Percent chnge in expression of most undnt micrornas in orts of GFM compred to PFM. n = 3 for ech microrna. (d) Reltive expression of Dicer nd Drosh in orts of GFM nd PFM. n = 3 for ech group. ns: not significnt y independent smple t-test. Dt shown s men nd error r represents s.e.m. Dicer Drosh
/ (% of PFM) Reltive expression of mirs y RTqPCR (% of PFM) Reltive expression of mirs y ncounter ssy (% of PFM) Reltive expression of mir-24 y ncounter ssy (% of PFM) Reltive expression of mir- 24 y RTqPCR (% of PFM) 15 1 5 ncounter ssy (n = 3) c 15 1 5 RTqPCR (n = 4) mir-29 Actin (et) mir-29 Actin (et) d 2 ncounter ssy 15 1 5 mir-24 mir-9 mir-148 mir-23 mir-145 e 2 RTqPCR 15 1 5 P =.6 Low High mir-24 mir-9 mir-148 mir-23 mir-145 f 3 (n = 4) 2 1 PFM GFM Hert Liver Muscle Aort Lung Supplementry Fig. 2. MiR-24 is downregulted in orts of GFM. () Het mp of ncounter rry showing microrna expression pttern normlized to cndidte micrornas, commonly used control mrnas, nd glol microrna expression (mmu-t-micrornas). Ech row represents microrna. Dt were clustered y pirwise complete linkge sed on Person correltion. Dt shows tht using mir-29 (red sterisk) s the internl normliztion control produced microrna expression profile tht most closely resemled the expression profile when using glol microrna (Totl mirna) s the normliztion control. (-c) mir-24 expression mesured y ncounter rry () nd qpcr (c) in orts of GFM nd PFM, normlized to microrna-29 nd -Actin s internl controls. p <.5 vs. PFM. (d) Expression of mir-24, mir-9, mir-148, mir-23 nd mir-145 mesured y ncounter ssy in orts of GFM nd PFM normlized to glol mirna. (e) Expression of mir-24, mir-9, mir-148, mir-23 nd mir-145 y qpcr in orts of GFM nd PFM normlized to mir-29. p <.5 vs. PFM. The selected micrornas represent down-regulted (mir-24, mir-9 nd mir-148), n upregulted (mir-23), nd highly expressed microrna with no chnge in expression (mir-145). (f) expression in hert, liver, ort, skeletl muscle, nd lungs of GFM nd PFM. n = 4 for ech group, p <.5 vs. PFM. Independent smple t-test ws used. Dt shown s men nd error r represents s.e.m. 2
vwf L mir-24 L Supplementry Fig. 3. mir-24 is expressed in vsculr endothelium nd tunic medi. Immunofluorescence for mir-24 in mouse orts. vwf: von Willernd fctor. L: lumen. (mgnifiction 63, Scle r; 2 m). 3
Control mir-24-i (2nM) mir-24-i (1nM) / (% of control) 11 HUVECs 24h c 3 25 85 6 35 11KD 2 15 1 5 1 24 48 72 Time (h) SC mir-24-m 1nM SC 2nM 1nM mir-24-i Supplementry Fig. 4. mir-24 regultes expression in endothelil cells. () mir-24 mimic (mir-24-m) suppresses expression in HUVECs in time dependent mnner. n = 3 independent experiments. p <.5 vs. SC. () mir-24 inhiitor (mir-24-i) upregultes expression in HUVECs. SC: scrmled control microrna. Representtive of 3 independent experiments. (c) Quntifiction of expression in. n = 3. p <.5 vs. SC. Independent smple t- test ws used. Dt shown s men nd error r represents s.e.m. 4
Body weight (g) Cholesterol (mg/dl) 4 R 1 5 N P D AB N P D -A B 3 1 N P D -A B -R 2 NPD NPD-AB NPD-AB-R 5 1 Weeks 3 6 9 12 15 18 To t l C h o le s t e ro l H D L C h o le s t e ro l Supplementry Fig. 5. Orl rod-spectrum ntiiotics slightly decrese ody weight nd do not ffect plsm lipids. () Effect of rod-spectrum ntiiotics on mouse ody weight. Arrowheds indicte isoltion of mice from the colony nd initition of ntiiotics in drinking wter (AB), nd discontinution of ntiiotics together with co-hittion of the mice with littermtes not on ntiiotics (AB-R). n = 6 for NPD nd NPD-AB; n = 3 for NPD-AB-R. () Effect of ntiiotics on totl cholesterol nd HDL-Cholesterol. n = 6 for NPD nd NPD-AB; n = 3 for NPD-AB-R. NPD: norml pellet diet; AB: Antiiotics; R: stoppge of ntiiotics + co-hittion. Dt shown s men nd error r represents s.e.m. 5
Fecl microiot lod (corrected to fecl mss, % of NPD) 1 5 1 5 1.. 5. N P D N P D -A B Supplementry Fig. 6. Orl rod-spectrum ntiiotics suppress fecl microil lod. Fecl microil lod ws mesured y qpcr of cteril 16S RNA in mice on norml pellet diet (NPD) or mice on NPD receiving rod-spectrum ntiiotics for 6 weeks (NPD-AB). n = 5 in ech group. P <.5 vs. NPD y independent smple t-test. Dt is shown s men normlized to NPD nd error r represents s.e.m. 6
SC+Ad-LcZ mir-24+ad-lcz mir-24+ad- mir-24 + Ad- mir-24 + Ad-LcZ mir-24/rnu6 (% of SC+Ad-LcZ) SC + Ad-LcZ 6 4 2 Supplementry Fig. 7. Ad- rescues endothelil expression suppressed y mir-24 mimic ex vivo. () mir-24 expression in explnted mouse orts trnsfected with mir-24 mimic, followed y infection with Ad or AdLcZ. n = 4 ortic rings from 3 mice. p <.5 vs. SC + AdLcZ y independent smple t-test. () Effect of mir-24 mimic nd/ Ad or AdLcZ on expression of endothelil in mouse orts. Arrowheds indicte endothelil immunostining for (Mgnifiction 1, Scle r; 2 m). SC: scrmled control microrna; Ad: denovirus encoding ; AdLcZ: control denovirus encoding E. Coli LcZ gene. Dt shown s men nd error r represents s.e.m. 7
NPD HFD HFD-AB HFD-AB-R HFD-AB-R Endothelil p-stt3 (% of NPD) HFD-AB mir-24/rnu6 (% of Ad-LcZ) HFD NPD DAPI Lmin-AF Stt3 11KD vwf p-stt3 Merge 5 4 3 2 1 c 15 A d -L c Z A d -D N S t t 3 1 5 Supplementry Fig. 8. Endothelil Stt3 signling inhiits mir-24 expression, is suppressed y HFD, nd rescued y rod-spectrum ntiiotics. () Adenovirl medited overexpression of dominnt negtive Stt3 (Ad-DNStt3) upregultes mir-24 expression in HUVECs. n = 4 in ech groups. p <.5 vs. AdLcZ. () Immunofluorescence stining (mgnifiction 63, Scle r; 2 m) of mouse ortic sections showing downregultion of endothelil phospho-stt3 y HFD feeding, nd rescue of endothelil STAT3 signling with suppression of gut microiot with rod-spectrum ntiiotics. Effect of ntiiotics on endothelil phospho-stt3 ws reversed with stoppge of ntiiotics. Lmin-AF: lminr utofluorescence. (c) Quntifiction of endothelil p-stt3 immunostining in. NPD: n = 5, HFD: n = 5, HFD-AB: n = 5 nd HFD-AB-R: n = 3. p <.5 vs. NPD, P <.5 vs. HFD. HFD: High-ft diet; NPD: norml pellet diet; AB: Antiiotics; R: stoppge of ntiiotics + co-hittion. Independent smple t-test ws used. Dt shown s men nd error r represents s.e.m. 8
N P D -S C H F D -S C H F D -2 4 -I N P D -S C H F D -S C H F D -2 4 -I Adiposity index Blood glucose (mg/dl) N P D -S C H F D -S C H F D -2 4 -I N P D -S C H F D -S C H F D -2 4 -I mir-24/ Body weight (g) 4 5 3 4 3 2 2 1 1 c 1 8 d 1 5 ns 6 1 4 5 2 Supplementry Fig. 9. Systemic inhiition of mir-24 does not ffect ody weight, diposity, nd lood glucose. () mir-24-i suppresses mir-24 expression in mouse orts. (-d) mir-24-i does not ffect ody weight (), diposity (c), nd fsting lood glucose (d). p <.5 vs. NPD, P <.5 vs. HFD. n = 4 for NPD-SC, n = 6 for HFD-SC nd HFD-24-I. NPD: norml pellet diet; HFD: highft diet; SC: scrmled control microrna. Independent smple t-test ws used. Dt shown s men nd error r represents s.e.m. 9
mir-24/rnu6 (% of NPD) Trpm3 (% of NPD) mir-24/rnu6 (% of NPD) c d 1 5 1 5 1 5 11KD p-stt3 82KD Stt3 82KD NPD NPD-AB 1 5 N PD N PD - A B 1 5 N PD N PD - A B 1 5 N PD ns N PD - A B - R Supplementry Fig. 1. Serum of mice on rod-spectrum ntiiotics promotes endothelil Stt3 signling nd, nd inhiits endothelil mir-24 expression. (-c) Serum of mice on rod-spectrum ntiiotics upregultes nd phospho-stt3 (), nd downregultes mir-24 nd Trpm3 (, c) in HUVECs. (d) Serum from mice in which ntiiotics were discontinued does not suppress mir-24 in HUVECs. n = 3 independent experiments, serum ws pooled from NPD (n = 6) nd NPD-AB (n = 4) nd NPD-AB-R (n = 3) mice. P <.5 vs. NPD. NPD: norml pellet diet; AB: Antiiotics; R: stoppge of ntiiotics + co-hittion. Independent smple t-test ws used. Dt shown s men nd error r represents s.e.m. 1
N PD H F D H F D - A B H F D - A B - S C F A N PD H F D H F D - A B H F D - A B - S C F A % Contrction (PE 1-6 M) mir-24 1 8 6 4 5 4 3 Mture mir-24 Pri- nd PremiR-24 2 N P D H F D H F D -A B H F D -A B -S C F A PE 1 9 8 7 6 5 2 1 log[m]a c 1 11KD HUVECs 24h.1 1 2.5 5. SCFAs(mM) (Ace., Prop. nd Buty.) d 11KD MASMCs 24h.1.5 1. 1 2.5 5.mM SCFAs(mM) (Ace., Prop. nd Buty.) Supplementry Fig. 11. Short-chin ftty cids (SCFAs) do not negte the effect of ntiiotics on endothelium-dependent vsorelxtion nd ortic mir-24 expression, nd do not ffect expression in endothelil or smooth muscle cells. (-c) Supplementtion of drinking wter with SCFAs [sodium cette (9mM) nd sodium utyrte (6mM)] does not negte () improvement in endothelium-dependent vsorelxtion nd () downregultion of mture nd precursor mir-24 in mouse orts induced y suppression of gut microiome y ntiiotics in HFD-fed mice. n represents numer of ortic rings in. NPD: n = 7, HFD: n = 9, HFD-AB: n = 12, HFD-AB-SCFA: n = 18. p <.5 vs. NPD. p <.5 vs. HFD. PE: phenylephrine; A: cetylcholine. (c-d) Mixture of SCFAs does not ffect 1 expression in HUVECs (c) nd mouse ortic smooth muscle cells (MASMCs) (d). Ace: cette; Prop: propionte; Buty: utyrte. Independent smple t-test ws used. Dt shown s men nd error r represents s.e.m. 11
% Contrction (PE 1-6 M) % Contrction (PE 1-6 M) 1 Fig. 2h Fig. 2i Fig. 2j Fig. 2k Fig. 2l 1 1 1 1 8 8 8 8 8 6 4 2 N P D N P D -A B N P D -A B -R 6 4 2 V E -C d -C r e V E -C d -C r e -A B 6 4 2 S I R T 1 fl/fl S ir t 1 fl/fl -A B 1 PE 9 8 7 6 5 PE 1 9 8 7 6 5 PE 1 9 8 7 6 5 PE 1 9 8 7 6 5 PE 1 9 8 7 6 5 log[m]snp log[m]snp log[m]snp log[m]snp log[m]snp 6 4 2 e S i r t 1 -/- e S i r t 1 -/- -A B 6 4 2 S C + A d -L c Z m ir - 2 4 + A d -L c Z m ir - 2 4 + A d -S ir t 1 Fig. 3h &i Fig. 3j Fig. 4c & d Fig. 4h Fig. 4j Suppl. Fig. 11 1 1 1 1 1 1 8 8 8 8 8 8 6 6 6 6 6 6 4 4 4 4 4 4 2 N P D -S C H F D H F D -A B H F D -A B -R 2 H F D H F D -A B 2 N P D -S C N P D -2 4 - I H F D -S C H F D -2 4 -I 2 N P D -S C N P D -2 4 - I H F D -S C H F D -2 4 -I 2 N P D -S C H F D -S C H F D -2 4 -I 2 N P D H F D H F D -A B H F D -A B -S C F A PE 1 9 8 7 6 5 PE 1 9 8 7 6 5 PE 1 9 8 7 6 5 PE 1 9 8 7 6 5 PE 1 9 8 7 6 5 log[m]snp log[m]snp log[m]snp log[m]snp log[m]snp 1 9 8 7 6 5 log[m]snp Supplementry Fig. 12. Sodium nitroprusside (SNP)-induced endothelium-independent vsorelxtion of the ort is not regulted y the gut microiome or mir-24. Dt corresponds to experiments done for the indicted figures in the mnuscript. 12
NPD-AB NPD NPD-SC HFD-SC HFD-24-I NPD HFD HFD-AB HFD-AB-R NPD HFD HFD-AB HFD-AB-R NPD HFD HFD-AB HFD-AB-R NPD-SC HFD-SC HFD-24-I Fig. 1e Fig. 3f Fig. 4e SC F4/8 11KD 11KD 11KD 12 24 36 48 72 mir-24-m Fig. 2d 12 24 36 48 72 82KD P-Stt3 Suppl. Fig. 8 P-Stt3 P-eNOS 11KD 82KD enos 11KD Fig. 4 AD-LcZ Ad-DNStt3 11KD 25KD 15KD 1KD Suppl. Fig. 1 Suppl. Fig. 11c 11KD 11KD HUVECs NPD-AB-R NPD NPD-AB p-stt3 82KD.1 1 2.5 5. SCFAs(mM) (Ace., Prop. nd Buty.) Suppl. Fig. 11d 11KD Stt3 82KD SCFAs(mM).1.5 1. 1 2.5 5.mM (Ace., Prop. nd Buty.) Supplementry Fig. 13. Originl immunolots for indicted figures. 13
Supplementry Tle 1. Sequence of primers, mture micrornas, detection proes nd micrornamimic/inhiitor used in the study. mrna Primer sequence Forwrd Reverse (mouse) 5 -AAT GCT GGC CTA ATA GAC TTG CA-3 5 -CCG TGG AAT ATG TAA CGA TTT GG-3 Trpm3 (mouse) 5 -ACC CCG TCA AGT AGT G-3 5 -CCC CAA AGT TGG CGT-3 TRPM3(humn) 5 -CGC AGC TGG AAG ACC TTA TC-3 5 -AAG CTG CTC TGA CGG ACA AT-3 (mouse) 5 -GGC AAA TTC AAC GGC ACA GT-3 5 -CGC TCC TGG AAG ATG GTG AT-3 GAPDH (humn) 5 -ATG ACA TCA AGA AGG TGG TG -3 5 -CAT ACC AGG AAA TGA GCT TG-3 enos (mouse) 5 -GAA GGG AAG TGC AGC AAA GG-3 5 -CAG AGA TCT TCA CTG CAT TGG CTA-3 Drosh (mouse) 5 -GGA CCA TCA CGA AGG ACA CT-3 5 -CAC GGG TCT CTT GGT TTT GT-3 Dicer (mouse) 5 -ACC AAG TGA TCC GTT TAC GC-3 5 -CAA CCG TAC ACT GTC CAT CG-3 microrna Mture microrna sequence mir-24-5p mir-29 mir-145-5p 5'-UUC CCU UUG UCA UCC UAU GCC U-3' 5'-UAG CAC CAU UUG AAA UCA GUG UU-3' 5'-GUC CAG UUU UCC CAG GAA UCC CU-3' mir-148-3p 5'-UCA GUG CAC UAC AGA ACU UUG U-3' mir-23-3p mir-9-5p 5'-AUC ACA UUG CCA GGG AUU ACC-3' 5'-UCU UUG GUU AUC UAG CUG UAU GA-3' microrna primers were procured from Qunt Bioscience. miscript Precursor ssy primer which detects stem-loop in oth pri- nd pre- mir-24 ws procured from QIAGEN. Modultors Sequence SC 5 -ACG TCT ATA CGC CCA- 3 mir-24-mimic mir-24-inhiitor mir-24 in-situ hyridiztion proe Scrmled in-situ hyridiztion proe 5'-UUC CCU UUG UCA UCC UAU GCC U-3' 5 -AGG ATG ACA AAG GGA-3 5 -Dig-N-AGG CAT AGG ATG ACA AAG GGA A-N-Dig-3 5 -Dig-N-GTG TAA CAC GTC TAT ACG CCC A-N-Dig-3 14