E10.5 E18.5 P2 10w 83w NF1 HF1. Sham ISO. Bmi1. H3K9me3. Lung weight (g)
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1 Myociyte cross-sectional Relative mrna levels Relative levels Relative mrna levels Supplementary Figures and Legends a Ezh2 E1.5 E18.5 P2 1w 83w b Ezh2 p16 amhc b-actin P2 43w kd wt mouse hearts c Sham TAC p16 Sham ISO NF1 HF1 kd d Human hearts b-actin e kd fl fl ;NkxCre H3K9me3 f 9 6 fl fl ;NkxCre b-actin 3 ANP BNP b-mhc a-mhc g 6 h.5 Lung weight (g) area (mm) Supplementary Figure 1. Cardiac-specific expression in cardiogenesis and adulthood (a) qrt-pcr analysis of wild-type mice for total cardiac expression of Ezh2 and mrna in embryos (E) at 1.5 and 18.5 dpc, newborns at postnatal day 2 (P2), 1-week-old adults, and aged adults (83-week-old). Results are standardized to β- actin and are expressed as the fold-change of the indicated subpopulations compared with the E1.5 population. Data are means ±SD (n=12, p<.5; Student s t-test). (b) Representative western blots (WB) showing the expression of, Ezh2, p16 INK4a (p16), αmhc, and β-actin in heart cells from wild-type newborns at postnatal day 2 (P2) and aged adults (43-week-old). (c) Representative western blots showing and p16 INK4a (p16) expression in heart samples from wild-type mice subjected to TAC or chronic infusion with ISO (n = 4
2 mice per group), and from representative human patients with healthy hearts (NF; n = 8) and with heart failure (HF; n = 8) (d) qrt-pcr detection of mrna expression in in non-failing and failing human hearts. (e) Representative western blots showing the protein levels of, H3K9me3, and β-actin in total heart cells from 22-week-old fl ;NkxCre and f/ mice. (f) qrt-pcr detection of mrna expression of the indicated genes in heart samples from fl ;NkxCre and f/ control mice (means ±SD; n=1, p<.5; Student s t-test). (g) Myocyte cross-sectorial area in fl ;NkxCre and f/ hearts (means ±SD; n=9, p<.5; Student s t-test). (h) Lung weight in 27-week-old fl ;NkxCre and f/ control mice (means ±SD; n=1, p<.5; Student s t-test).
3 WHA HW/BW Masson p16 ARF Ezh2 p53 p21 HW/BW a b c IVSd (mm) LVIDd (mm) LVIDs (mm) LVPWs (mm) FS (%) EF (%) LVmass (mg) fl/+ fl/+ ;amhccre ws 6months ws 6months 7ws 6months 7ws 6months 7ws 6months 7ws 6months 3 7ws 6months d fl/+ fl/+ ;amhccre e fl/+ fl/+ ;amhccre f % Overall survival 1 fl/+ fl/+ ;amhccre n=12 n= Time (weeks) Supplementary Figure 2. Adult deficiency causes dilated cardiomyopathy (a) qrt-pcr analysis of, p16 INK4a, ARF, Ezh2, p21, and p53 mrna expression in total heart cells from fl ;αmhccre (-KO) mice. Data are standardized to actin levels, and are expressed relative to f/f (Control) mice (means ±SD; n=12, p<.1, p<.5; Student s t-test). (b) Heart-to-body-weight (HW/BW) ratio in 12-week-old fl ;αmhccre mice and f/f controls (means ±SD; n=12, p<.5; Student s t-test). (c) Echocardiographic measurements and physiological parameters in 7-week- and 6- month-old f/+ ;αmhccre heterozygotes and f/+ controls mice. IVSd, diastolic interventricle septal wall thickness; LVDd, diastolic left ventricle internal dimension; LVDs, systolic left ventricle internal dimension; LVPWd, diastolic left ventricle posterior wall thickness; FS, fractional shortening of left ventricle dimension; EF, ejection fraction; LVmass, left ventricle mass. Data are means ±SD; n=12, p<.1, p<.5; Student s t-test). (d) Heart-to-body-weight (HW/BW) ratio in 6-month-old f/+ ;αmhccre heterozygotes and f/+ control mice (means ±SD; n=1, p<.5; Student s t- test).
4 (e) Representative Masson's trichrome staining to detect fibrosis (bars, 4 μm) and WGA staining of left ventricle to outline cardiomyocytes (bars, 1 μm) in 6-monthold f/+ ;αmhccre heterozygotes and f/+ control mice. (f) Kaplan-Meier survival curve for f/+ ;αmhccre heterozygotes and f/+ littermate mice (means ±SD; p<.1; Student s t-test).
5 % Engraftment in PB cells Wall thickness (mm) FS (%) Myociyte cross-sectional Myociyte cross-sectional a kd 37 i tg i tg MLC2 b 6 15 hearts H3K9me3 b-actin area (mm) TAC i tg i tg MLC2v c fl fl ;amhccre d fl fl ;amhccre e area (mm) fl fl ;amhccre IgG Tgf-b ab IgG Tgf-b ab IgG Tgf-b ab f % 45.2% 2 CD45.1 CD45.1 no-dcm no-dcm no-dcm DCM high CD45.1 CD45.1 CD45.1 CD45.2 Supplementary Figure 3. Effect of Tgf- inhibition in DCM mice (a) Representative western blots (WB) showing the protein levels of, H3K9me3, and β-actin in total heart cells from doxocycline-induced 12-week-old i tg/tg ;MLC2 tg/+ (i tg ;MLC2) mice and i tg/tg ;MLC2 +/+ (ibmi tg ) control littermates. (b) Quantification of myocyte cross-sectional area in nontransgenic and transgenic mice subjected to TAC surgery or sham (means ± s.d. n = 5 mice per group). (c-e) Left ventricular wall thickness (c), fractional shortening (d), and of myocyte cross-sectional area (e) in 12-week-old fl/fl ;p16 INK4a-/-, fl/fl ; MHC-Cre and fl ; MHC-Cre;p16 -/- mice (means ± s.d. n = 8 mice per group; p<.5). (f) Blood chimerism was analyzed in parabiotic pairs as indicated by measuring the frequency of donor-derived blood cells from one partner (CD ) to the other (means ± SD; n=6; p<.5).
6 Supplementary Table 1 Primer sequences used for genotyping cko- fl/fl mice Fw LOXP1 AGAGAATCCAGCTGTCCAGTG Rv LOXP1 CCTGGACATCACAAATAGGACA αmhc TM -Cre tg/+ mice Fw Tg alfa Mhc Cre Ert 2 AGGTGGACCTGATCATGGAG Rv Tg alfa Mhc Cre Ert 2 ATACCGGAGATCATGCAAGC Fw Int Pos Control CTAGGCCACAGAATTGAAAGATCT Rv Int PosControl GTAGGTGGAAATTCTAGCATCATCC MLC2-rRTA tg/+ mice RTTA FW CAGAGCCAGCCTTCTTATTC RTTA RV ACGTCCTCGCCGTCTAAGTG Fw 5 Arm 17pb 5'-GGAGGGGAGTGTTGCAATACC p16 INK4a-/- mice FW LOX P1 CCTGACTATGGTAGTAAAGTGG FW LOX P2 ACGTGTATGCCACCCTGACC αmhc-cre tg/+ mice Fw MHC GCGGTCTGGCAGTAAAAACTATC Rv MHC GTGAAACAGCATTGCTGTCACTT Fw Int Pos Control CTAGGCCACAGAATTGAAAGATCT Rv Int PosControl GTAGGTGGAAATTCTAGCATCATCC Nkx2.5-Cre mice NKX 2,5 ANTISENSE GCGCACTCACTTTAATGGGAAGAG NKX 2,5 SENSE GCCCTGTCCCTCAGATTTCACACC NKX 2,5CRE GATGACTCTGGTCAGAGATACCTG
7 Primer and probe sequences used in qrt-pcr Fw ACCTGCTGCTGGCCCCTTC Rv GACTCCTTGATGAAGGTGCCC Fw Ezh2 GCCCACCTCGGAAATTTCCTGC Rv Ezh2 CAGAGCACCTGGGAGCTGCTG Fw mbmi1 int CAGCAATGACTGTGATGC Rv mbmi1 int CTCCAGCATTCGTCAGTC Fw p16 E1 CGAACTCTTTCGGTCGTACCC Rv p16 E2 TTGAGCAGAAGAGCTGCTACG Fw1 mp15 qpcr AGATCCCAACGCCCTGAACCG Rv1 mp15 qpcr TGCTCTTCAGCCAAGTCTACC Fw ARF TTTGAGGTTCGTGTTTGTGC Rv ARF TTCTTTTGCGGGGGAGAG Fw Myh7 CAACACCAGCCACCCTCTAT Rv Myh7 GAGAAGGTTTGGGTCTCGTG Fw Tgfb TACCCTGGAAGTCCTGCTTG Rv Tgfb AACTGCCAGGGATGGAAAAT FW E1 p53 CCTGAGACCTGCCCCTGTC RV E2 p53 GGCACACTTCTCACATCCGGC FW p21 GCTTTCACTACACATTTGCCC RV p21 CCACATCAAACGCCTTGCGG FW ANP GAGGAGCAAGAGACTAAGGGC RV ANP CAGGCCTGCAGGTCCCTGC FW BNP CATCAATTACCTGCCCCTACC RV BNP CAAGGGTGGAGGTCATTGGG FW bmhc TCCCTCGGGAAACGGGACCC RV bmhc CCTGCCCAAGGTTGGAGTCG FW amhc CGCGCTCTGCATCAGTGACG RV amhc GCCCTTGCATCTCCCACGG
8 qpcr primers used for ChIP analysis Tgfb3 FW4 PROM CyclinA2 FW3PROM RV3PROM FW4PROM IL1 FW4 PROM Gbe1 FW4 PROM Gmnn TTTAATTTCCCTTGTAGACAGCC TGTGCTAGCTTTTCAGTCGGG TTCCCTTGTAGACAGCCTTCC ACCTAAATCGGATTAGCAACCC GAGACTTCTTGAACTTGCCGGG GGAAGAGAGGTGATCGGGC CGAAGAAAAGCAACAGAAGCCC CTTTAGGAGACCGCAGTCCG AACCTTCACAGAGTGGACCCC CAGAAAACTAAACTCTTGACTACC CCTGACCTGCCTGCAAATGGC CATCTAGCTACACATCATAGGG AATCAAGGCTTTTGGGAGGTCC AGTTATATGAACTAAACACCCC AACACAGAACTGCTCTCTCCCC GGAATGATTTTGGATAGACTTCCG TGCGAAAGCCTGTGGTTTGC TTTGGACCTCATAAACAACCAC TGTTCCCCTTTCCACAGCCC TTTCCTTACACAGCCTGGGGG AGAGATTCAGTGGCTGCCAGC AGAATCTTCAACAGCTCTGCC CAGGTGTCTTTGTTAATGGCCC CCATACAATGAAAAACCCTGACC ATCCGCCTGCCTCTGCCTCC CCTGTCAATCCAAACTTGAGCC CCAATGTTCCTCTAAAGTCCC TCAGTGAATGCCACATTTAGGG TTTTGTGGTCGAATTTCTTGGG TGTTAGCTGTTGCTGCTACCC ATCATGAAATCTTGCACCAGCC AAATAATCATTTGGGTGGAGGG GCTCGCACCTACAAAGCCCG TGTAAACAAAGTGAGGGCCAGG ACTTGGCTCCTTTGCTCCCC GGCTGACTCAGATGACTGGG GCAGGTGAGTCACAGCACCG TTGACTAGGATCCGCCTGCC
9 FW4 PROM P16INK4a FW4 PROM TCTTTCTTCCCTGAAATCTGGC TCTTCACCTTCTCTGCTCTCC TGATAAAACTGAGTTAGAAAGGC GAAATTTATGACAGCTTTATTCCC AACCATTTCAAGCTTGTAGAGG CAGAGAGATGTGGTGTGATAGC AGCTACTGACCTAGAATTGGG CTAGGAAAGTAGTACTTTGGGC CATACAAAGGTGATCCCTGCC TCACAGAATGCCTAGAAGGCC
Tcf21 MCM ; R26 mtmg Sham GFP Col 1/3 TAC 8W TAC 2W. Postn MCM ; R26 mtmg Sham GFP Col 1/3 TAC 8W TAC 2W
A Tcf21 MCM ; R26 mtmg Sham GFP Col 1/3 Tcf21 MCM ; R26 mtmg TAC 2W Tcf21 MCM ; R26 mtmg TAC 8W B Postn MCM ; R26 mtmg Sham GFP Col 1/3 Postn MCM ; R26 mtmg TAC 2W Postn MCM ; R26 mtmg TAC 8W Supplementary
c Ischemia (30 min) Reperfusion (8 w) Supplementary Figure bp 300 bp Ischemia (30 min) Reperfusion (4 h) Dox 20 mg/kg i.p.
a Marker Ripk3 +/ 5 bp 3 bp b Ischemia (3 min) Reperfusion (4 h) d 2 mg/kg i.p. 1 w 5 w Sacrifice for IF size A subset for echocardiography and morphological analysis c Ischemia (3 min) Reperfusion (8
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Kanazawa, et al. Supplementary figure legends Supplementary Figure 1 DS rats had congestive heart failure. (A) DR and DS rat hearts. (B) QRT-PCR analysis of BNP mrna expression in DR and DS rat left ventricles
Postn MCM Smad2 fl/fl Postn MCM Smad3 fl/fl Postn MCM Smad2/3 fl/fl. Postn MCM. Tgfbr1/2 fl/fl TAC
A Smad2 fl/fl Smad3 fl/fl Smad2/3 fl/fl Tgfbr1/2 fl/fl 1. mm B Tcf21 MCM Tcf21 MCM Smad3 fl/fl Tcf21 MCM Smad2/3 fl/fl Tcf21 MCM Tgfbr1/2 fl/fl αmhc MCM C 1. mm 1. mm D Smad2 fl/fl Smad3 fl/fl Smad2/3
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a c e doi:10.1038/nature10407 b d f Supplementary Figure 1. SERCA2a complex analysis. (a) Two-dimensional SDS-PAGE gels of SERCA2a complexes. A silver-stained SDSPAGE gel is shown, which reveals a 12 kda
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Combination index (CI) Supplementary Figure 1 2. 1.5 1. Ishikawa AN3CA Nou-1 Hec-18.5...2.4.6.8 1. Fraction affected (Fa) Supplementary Figure 1. The synergistic effect of PARP inhibitor and PI3K inhibitor
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Supplemental Figure I Kl ( mmol/l)-induced Force orta M (mn) 1 (mn) 1 Supplemental Figure I. Kl-induced contractions. and, Kl ( mmol/l)-induced contractions of the aorta () and those of mesenteric arteries
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1 SUPPLEMENTARY INFORMATION Mutations in the NOTCH pathway regulator MIB1 cause left ventricular noncompaction cardiomyopathy Guillermo Luxán, Jesús C. Casanova, Beatriz Martínez-Poveda, Belén Prados,
Supplement Material. chain (MLC2v) promoter to generate cardiac-specific MKK4 knockout mice (referred to
Supplement Material Materials and Methods Generation of MKK Mutant Mice The mkk-flox mice (referred to as ) were previously generated and characterised. mice were mated with mice expressing Cre under a
X P. Supplementary Figure 1. Nature Medicine: doi: /nm Nilotinib LSK LT-HSC. Cytoplasm. Cytoplasm. Nucleus. Nucleus
a b c Supplementary Figure 1 c-kit-apc-eflu780 Lin-FITC Flt3-Linc-Kit-APC-eflu780 LSK Sca-1-PE-Cy7 d e f CD48-APC LT-HSC CD150-PerCP-cy5.5 g h i j Cytoplasm RCC1 X Exp 5 mir 126 SPRED1 SPRED1 RAN P SPRED1
Supplemental Figure 1
Supplemental Figure 1 1a 1c PD-1 MFI fold change 6 5 4 3 2 1 IL-1α IL-2 IL-4 IL-6 IL-1 IL-12 IL-13 IL-15 IL-17 IL-18 IL-21 IL-23 IFN-α Mut Human PD-1 promoter SBE-D 5 -GTCTG- -1.2kb SBE-P -CAGAC- -1.kb
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J Jpn Coll Angiol, 2009, 49:
Online publication October 6, 2009 48 2 20 J Jpn Coll Angiol, 2009, 49: 293 297 atrial natriuretic peptide, brain natriuretic peptide, guanylyl cyclase-a receptor, cardiac remodeling, cardiac hypertrophy
Supplementary Information and Figure legends
Supplementary Information and Figure legends Table S1. Primers for quantitative RT-PCR Target Sequence (5 -> 3 ) Target Sequence (5 -> 3 ) DAB2IP F:TGGACGATGTGCTCTATGCC R:GGATGGTGATGGTTTGGTAG Snail F:CCTCCCTGTCAGATGAGGAC
Supplementary Information File
Supplementary Information File Supplementary Table 1. List of synthesized sirna sequences for target genes sirna Species Sequence Ctrl sirna mouse sense 5 -UUCUCCGAACGUGUCACGUTT-3 Antisense 5 -ACGUGACACGUUCGGAGAATT-3
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