supplementary information - Nature Research · are S.E.M.). Uncropped images of blots are shown in Supplementary Information, Fig.S6. Supplemental Figure 2 luciferase Dyrk1A 3’UTR
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s u p p l e m e n ta ry i n f o r m at i o n
www.nature.com/naturecellbiology 1
DOI: 10.1038/ncb2126
Figure S1 Calcineurin/NFAT-dependent transcriptional induction of miR-199b. (a) Dynamin-1 (Dnm1) expression, the host gene of miR-199b, remains unaltered in cardiac disease. Analysis of expression of Dnm1 transcripts by real time PCR in cardiac tissue of two experimental models of heart failure: transgenic mice engineered to overexpress a constitutively activated mutant of calcineurin in the postnatal myocardium (MHC-CnA) and mice subjected to pressure overload by transverse aortic constriction (TAC). (b) Luciferase reporters encompassing 1 kb upstream regions of the miR-199b gene related to the genomic location of miR-199b, located on the opposite strand of the
mouse dynamin 1 (Dnm1) gene on chromosome 2 in the murine genome. Reporters were tested for calcineurin/NFAT responsiveness in transient co-transfection experiments with vectors expressing an active calcineurin mutant and NFATc2. (c) Sequence alignment and evolutionary conservation between different species of three candidate NFAT sites located within -3.5 kb relative to the mmu-miR-199b gene. Sequences were compared to a minimal consensus NFAT binding motif (A/TGGAAA or TTTCCT/A) and site-directed nucleotide mutations used in luciferase reporters are shown in blue. Data are presented as mean (error bars are S.E.M.).
Figure S2 In vitro validation of miR-199b target mRNAs. (a) mRNA levels of predicted miR-199b target genes were assessed by standard RT-PCR. L7 served as a loading control. (b) Western blot validation of four putative miR-199b target genes in Dox inducible miR-199b clone. Dyrk1a, dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1a; Myl6b, myosin, light polypeptide 6B; GPCR5A, G protein-coupled receptor, family C, group 5, member A; AP1G1, adaptor protein complex AP-1, gamma 1 subunit. GAPDH, glyceraldehyde-3-phosphate dehydrogenase,
was used as a loading control. (c) Activity of luciferase reporter construct harboring the 3’UTR of Dyrk1a after transfection of indicated synthetic precursor microRNAs. (d) Activity assay of luciferase reporter constructs harboring an intact (pMIR-Dyrk1a) or mutated Dyrk1a 3’UTR (pMIR-Dyrk1a mut) after doxycylin-induced miR-199b-5p expression in clones from (b). *P < 0.05 vs corresponding control group (error bars are S.E.M.). Uncropped images of blots are shown in Supplementary Information, Fig.S6.
Figure S3 miR-199b overexpression sensitizes the myocardium to cardiac hypertrophy. (a) Gravimetric analysis of corrected heart weights in MHC-199b mice at 6 months of age. (b) Real time PCR analysis of transcript abundance for fetal marker genes and rcan1-4 in hearts from MHC-199b mice at 6 months of age. (c) Quantification of Rnu6-2 corrected Northern blot miR-199b-5p signals from non-transgenic (nTg), single transgenic (MHC-199b or MHC-CnA) and double transgenic mice (DTg). (d) Quantification of GAPDH corrected Western blot Dyrk1A signals from nTg, MHC-199b, MHC-CnA or DTg mice from Fig. 3d. (e) Quantification of
Rnu6-2 corrected Northern blot miR-199b-5p signals from non-transgenic (nTg) and MHC-199b transgenic mice subjected to sham or pressure overload by TAC (1 week). (f) Western blot analysis and quantification of Dyrk1a and GAPDH in non-transgenic (nTg) and MHC-199b transgenic mice subjected to sham or TAC. (g) Real time PCR analysis of transcript abundance for indicated fetal marker genes in hearts from nTg or MHC-199b mice, subjected to 1 week sham or TAC. *P < 0.05 vs corresponding control group; #P < 0.05 vs experimental group (error bars are S.E.M.). Uncropped images of blots are shown in Supplementary Information, Fig.S6.
Supplemental Figure 3.
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Figure S4 Dyrk1A haploinsufficiency sensitizes the myocardium to cardiac hypertrophy. (a) Western blot analysis and quantification of Dyrk1A and GAPDH in hearts from Dyrk1A+/+ and Dyrk1a haploinsufficient mice (Dyrk1A+/-) subjected to sham or TAC. (b) Real time PCR analysis of transcript abundance for indicated fetal marker genes in hearts from Dyrk1A+/+ and Dyrk1a+/- mice subjected to sham or TAC surgery for 1 week. (c) Gravimetric analysis of corrected heart weights in 3 week old Dyrk1a+/+, MHC-CnA/Dyrk1a+/+, Dyrk1a+/- and MHC-CnA/Dyrk1a+/- mice. (d) Western blot analysis and quantification of Dyrk1A and GAPDH in hearts from Dyrk1a+/+, MHC-CnA/
Dyrk1a+/+, Dyrk1a+/- and MHC-CnA/Dyrk1a+/- mice. (e) Representative image of whole hearts (top panels), H&E- (mid panels) or Sirius Red stained (lower panels) histological sections of hearts from Dyrk1a+/+, MHC-CnA/Dyrk1a+/+, Dyrk1a+/- and MHC-CnA/Dyrk1a+/- mice. (f) Real time PCR analysis of transcript abundance for fetal marker genes and rcan1-4 in hearts from Dyrk1a+/+, MHC-CnA/Dyrk1a+/+, Dyrk1a+/- and MHC-CnA/Dyrk1a+/- mice. Data are presented as mean ±S.E.M.; *P < 0.05 vs corresponding control group; #P < 0.05 vs experimental group (error bars are S.E.M.). Uncropped images of blots are shown in Supplementary Information, Fig.S6.
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Figure S5 Antagomir-mediated miR-199b silencing prevents and reverses cardiac remodeling and dysfunction. (a) Northern blot analysis of miR-199a-5p expression (encoded by mmu-miR-199a-1 on murine chromosome 9 and mmu-miR-199a-2 on murine chromosome 1) in hearts from mice treated with vehicle or antagomir-199b as a control for specificity of antagomir-199b, Rnu6-2 expression was used as loading control. (b) Western blot quantification of Dyrk1A and GAPDH in hearts from nTg or MHC-CnA mice treated with antagomir-199b. (c) Northern blot and Western blot analysis of miR-199b and Dyrk1A, respectively, in hearts from wildtype mice subjected to sham or TAC for 6 weeks (prevention study, p) and treated with antagomir-199b, the small RNA molecule U6 small nuclear (Rnu6-2) and GAPDH were used as loading controls, respectively. (d) Quantification of Rnu6-2 corrected Northern blot miR-199b-5p signals from mice in
(c). (e) Quantification of GAPDH corrected Western blot Dyrk1A signals from mice in (c). (f) Real time PCR analysis of transcript abundance for fetal marker genes and rcan1-4 in hearts from mice in (c). (g) Northern blot and Western blot analysis of miR-199b and Dyrk1A, respectively, in hearts from wildtype mice subjected to sham or TACr for 6 weeks (r, reveral study). Rnu6-2 and GAPDH were used as loading controls, respectively. (h) Quantification of Rnu6-2 corrected Northern blot miR-199b-5p signals from mice in (g). (i) Quantification of GAPDH corrected Western blot Dyrk1A signals from mice in (g). (j) Real time PCR analysis of transcript abundance for fetal marker genes and rcan1-4 in hearts from (g). *P < 0.05 vs corresponding control group; #P < 0.05 vs experimental group (error bars are S.E.M.). Uncropped images of blots are shown in Supplementary Information, Fig.S6.
Supplemental Figure 5
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Data are expressed as means ± SEM. IVSd, interventricular septal thickness at end-diastole; IVSs, interventricular septal thickness at end-systole; LVIDd, left ventricular internal dimension at end-diastole; LVIDs, left ventricular internal dimension at end-systole; LVPwd, left ventricular posterior wall thickness at end-diastole; LVPws, left ventricular posterior wall thickness at end-systole; FS, fractional shortening; LV, left ventricular; VcF, circumferential fiber shortening; E/A, Doppler E/A ratio. * indicates P<0.05 vs corresponding sham group; # indicates P<0.05 vs t=0, same group; § indicates P<0.05 vs vehicle treated group, same time point.
Data are expressed as means ± SEM. IVSd, interventricular septal thickness at end-diastole; IVSs, interventricular septal thickness at end-systole; LVIDd, left ventricular internal dimension at end-diastole; LVIDs, left ventricular internal dimension at end-systole; LVPwd, left ventricular posterior wall thickness at end-diastole; LVPws, left ventricular posterior wall thickness at end-systole; FS, fractional shortening; LV, left ventricular; VcF, circumferential fiber shortening; E/A, Doppler E/A ratio. * indicates P<0.05 vs corresponding sham group; # indicates P<0.05 vs t=0, same group; § indicates P<0.05 vs vehicle treated group, same time point.