Gallic acid prevents isoproterenol-induced cardiac hypertrophy and fibrosis through regulation of JNK2 signaling and Smad3 binding activity
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1 Gallic acid prevents isoproterenol-induced cardiac hypertrophy and fibrosis through regulation of JNK2 signaling and Smad3 binding activity Yuhee Ryu 1,+, Li Jin 1,2+, Hae Jin Kee 1,, Zhe Hao Piao 3, Jae Yeong Cho 1, Gwi Ran Kim 1, Sin Young Choi 1, Ming Quan Lin 1,4, and Myung Ho Jeong 1, 1 Heart Research Center of Chonnam National University Hospital, Gwangju , Republic of Korea. 2 Jilin Hospital Affiliated with Jilin University, Jilin, China 3 The Second Hospital of Jilin University, Changchun, China 4 Yanbian University Hospital, 1327 Juzi Road, Yanbian, Jilin 133, China Supplementary Data 1
2 Supplementary Table 1 Supplementary Table 1. Primers for reverse transcription polymerase chain reaction (RT-PCR) and chromatin immunoprecipitation (ChIP) Gene (mouse) ANP BNP Collagen I Fibronectin Alpha SMA GAPDH (mouse) Collagen type I (ChIP) F, forward; R, reverse. Primer sequence (5ʹ to 3ʹ) F: TGGAGGAGAAGATGCCGGTAGAAGAT R: AGCGAGCAGAGCCCTCAGTTTGCT F: CTGAAGGTGCTGTCCCAGAT R: GTTCTTTTGTGAGGCCTTGG F: GAGCGGAGAGTACTGGATCG R: GCTTCTTTTCCTTGGGGTTC F: GATGCACCGATTGTCAACAG R: TGATCAGCATGGACCACTTC F: ACTGGGACGACATGGAAAAG R: AGAGGCATAGAGGGACAGCA F: GCATGGCCTTCCGTGTTCCT R: CCCTGTTGCTGTAGCCGTATTCAT F: TGAGAAGTGGCAGAGGAGGT R: GACTGCCACATCAAGGGTCT 2
3 2. Supplementary Figures Legends Supplementary Figure 1. Effect of gallic acid on isoproterenol (ISP)-treated H9c2 cells. H9c2 cells were incubated with ISP I (1 µmol/l) in the presence or absence of gallic acid (1 µmol/l). (A) Cells were fixed with 4% paraformaldehyde and incubated with sarcomeric α-actinin (1:2). Nuclei were stained with DAPI. Merged images are shown. (B) Cell size was measured. (C) Protein lysates were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with the indicated antibodies. Representative western blots are shown. (D-F) Quantification of β-mhc, ANP, and BNP protein levels was performed using densitometry. Protein expression was normalized to GAPDH. Values are means ± SD of 3 independent experiments. P < 5 versus vehicle. # P < 5 and ## P < 1 versus ISP. Supplementary Figure 2. Echocardiographic parameters in ISP-infused mice. (A) Representative M-mode images of left ventricles in mice infused with ISP (1~3 day) as indicated. (B F) Left ventricular posterior wall thickness, interventricular septum thickness, left ventricular end-systolic dimension, left ventricular end-systolic dimension, and fractional shortening were measured after ISP infusion. P < 5, P < 1, and P < 1 versus control. (G) Heart weight to body weight ratio. Supplementary Figure 3. Expression of MAPK signaling in ISP-treated cardiac fibroblasts. Rat cardiac fibroblasts were serum-starved and incubated with ISP I (1 µmol/l) at indicated time. Proteins were analyzed by western blot with anti-p-erk1/2 (Thr 22/Thy24), ERK, p-jnk1/2 (S423/425), and JNK antibodies. β-actin was used as an internal control. Supplementary Figure 4. Expression of Smad3 in ISP-treated cardiac fibroblasts. Gallic acid administration was started 2 week before infusion of ISP (3 day) in mice; sham + vehicle, ISP + vehicle, and ISP + gallic acid group. Proteins were subjected to western blotting with anti-smad3 antibody. GAPDH was used as a loading control. Smad3 protein was quantified by densitometry. # P<5 versus ISP + vehicle. NS indicates not significant. Supplementary Figure 5. Expression of phosphorylated Smad3 in ISP-treated cardiac fibroblasts. Rat cardiac fibroblasts were serum-starved and incubated with ISP I (1 µmol/l) at indicated time. Proteins were analyzed by western blot with anti-psmad3 (S423/S425) and Smad3 antibodies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. 3
4 Supplementary Figure 1 A C Control + vehicle ISP + vehicle ISP + Gallic acid ISP Gallic acid β-mhc ANP BNP GAPDH E Relative ANP protein 2. ## F Relative BNP protein B Cell size (arbitrary unit) D Relative MHC protein 2. # ### ISP Gallic acid ## ISP Gallic acid ISP Gallic acid ISP Gallic acid
5 Supplementary Figure 2 A ISP d ISP 1 d ISP 2 d ISP 3 d B left ventricular posterior wall thickness (mm) C Interventricular septum thickness (mm) D left ventricular end-systolic dimension (mm) E left ventricular end-diastolic dimension (mm) F Fractional shortening (%) G Heart weight / body weight ratio n=12 n=2 n=2 n=13
6 Supplementary Figure 3 ISP Time (h) p-erk ERK p-jnk JNK β-actin Rat cardiac fibroblast
7 Supplementary Figure 4 Relative Smad3/GAPDH protein NS n=12 n=15 n=15 ISP Gallic acid #
8 Supplementary Figure 5 ISP (h) p-smad3 (S423/425) Smad3 GAPDH Cardiac fibroblast 54 kda 54 kda 38 kda
hemodynamic stress. A. Echocardiographic quantification of cardiac dimensions and function in
SUPPLEMENTAL FIGURE LEGENDS Supplemental Figure 1. Fbn1 C1039G/+ hearts display normal cardiac function in the absence of hemodynamic stress. A. Echocardiographic quantification of cardiac dimensions and
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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Myociyte cross-sectional Relative mrna levels Relative levels Relative mrna levels Supplementary Figures and Legends a 8 6 4 2 Ezh2 E1.5 E18.5 P2 1w 83w b Ezh2 p16 amhc b-actin P2 43w kd 37 86 16 wt mouse
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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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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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Supplementary Fig. S1. Schematic diagram of minigenome segments.
open reading frame 1565 (segment 5) 47 (-) 3 5 (+) 76 101 125 149 173 197 221 246 287 open reading frame 890 (segment 8) 60 (-) 3 5 (+) 172 Supplementary Fig. S1. Schematic diagram of minigenome segments.
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Supplemental Figure 1
Supplemental Figure 1 A S100A4: SFLGKRTDEAAFQKLMSNLDSNRDNEVDFQEYCVFLSCIAMMCNEFFEGFPDK Overlap: SF G DE KLM LD N D VDFQEY VFL I M N FF G PD S100A2: SFVGEKVDEEGLKKLMGSLDENSDQQVDFQEYAVFLALITVMCNDFFQGCPDR
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Supplementary Figure 1: (A) Whole aortic cross-sections stained with Hematoxylin and Eosin (H&E), 7 days after porcine-pancreatic-elastase (PPE)-induced AAA compared to untreated, healthy control aortas
(A) PCR primers (arrows) designed to distinguish wild type (P1+P2), targeted (P1+P2) and excised (P1+P3)14-
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[Billing Code: ] DEPARTMENT OF HEALTH AND HUMAN SERVICES. Office of the Secretary. Findings of Research Misconduct
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This document is scheduled to be published in the Federal Register on 06/03/2016 and available online at http://federalregister.gov/a/2016-13072, and on FDsys.gov [Billing Code: 4150-31] DEPARTMENT OF
Appendix Table of Contents. 1. Appendix Figure legends S1-S13 and Appendix Table S1 and S2. 2. Appendix Figures S1-S13
Appendix Table of Contents. Appendix Figure legends S-S3 and Appendix Table S and S. Appendix Figures S-S3 . Appendix Figure legends S-S3 and Appendix Table S and S Appendix Figure S. Western blot analysis
Gene expression was analyzed by real time PCR (SYBR GREEN PCR Master. Mix, Roche Applied Science) using specific oligonucleotides.
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Table S1. Primer sequences used for qrt-pcr. CACCATTGGCAATGAGCGGTTC AGGTCTTTGCGGATGTCCACGT ACTB AAGTCCATGTGCTGGCAGCACT ATCACCACTCCGAAGTCCGTCT LCOR
Table S1. Primer sequences used for qrt-pcr. ACTB LCOR KLF6 CTBP1 CDKN1A CDH1 ATF3 PLAU MMP9 TFPI2 CACCATTGGCAATGAGCGGTTC AGGTCTTTGCGGATGTCCACGT AAGTCCATGTGCTGGCAGCACT ATCACCACTCCGAAGTCCGTCT CGGCTGCAGGAAAGTTTACA
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a GPR120 GPR120 mrna/ppia mrna Arbitrary Units 150 100 50 Liver BAT iwat ewat mwat Ileum Colon b UCP1 mrna Fold induction 20 15 10 5 - camp camp SB202190 - - - H89 - - - - - GW7647 Supplementary Figure
Supplementary data Supplementary Figure 1 Supplementary Figure 2
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ZL ZDF ZDF + E2 *** Visceral (g) ZDF
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Supplementary Figures
Supplementary Figures Supplementary Figure 1 Correlation between LKB1 and YAP expression in human lung cancer samples. (a) Representative photos showing LKB1 and YAP immunohistochemical staining in human
Supplementary Figure 1. Western blot of hippocampal lysates from WT and Adcy1 KO mice demonstrates the specificity of the ADCY1 antibody.
ADCY1 13 kda β-actin 45 kda Supplementary Figure 1. Western blot of hippocampal lysates from and mice demonstrates the specificity of the ADCY1 antibody. a DHPG perk1/2 ERK1/2 Relative level min 1.6 *
5 -GGTAAAGCAGGTCTAGGTGGCTGACAGTCT-3. Cre-transgene allele was detected. by PCR using the primers; 5 -ACATGTTCAGGGATCGCCAG-3 and
Supplemental Information Methods PCR primers for mice genotyping The Gab1 flox allele can be distinguished from the wild type Gab1 allele by PCR on genomic DNAs extracted from tails, using the primer pair
Supplementary Figure 1: Co-localization of reconstituted L-PTC and dendritic cells
a CD11c Na + K + ATPase Na + K + ATPase CD11c x-y CD11c Na + K + ATPase Na + K + ATPase CD11c x-z c b x-y view BoNT NAPs CD11c BoNT CD11c NAPs BoNT NAPs CD11c 90 x-z view Apical Basolateral Supplementary
Figure 1. Effects of FGF21 on adipose tissue. (A) Representative histological. findings of epididymal adipose tissue (B) mrna expression of
SUPPLEMENTAL MATERIAL EN-12-2276 Figure 1. Effects of FGF21 on adipose tissue. (A) Representative histological findings of epididymal adipose tissue (B) mrna expression of adipocytokines in adipose tissue.
Supplementary Materials for
advances.sciencemag.org/cgi/content/full/1/9/e1500781/dc1 Supplementary Materials for pnaktide inhibits Na/K-ATPase reactive oxygen species amplification and attenuates adipogenesis Komal Sodhi, Kyle Maxwell,
Supplementary Figure 1. Repression of hepcidin expression in the liver of mice treated with
Supplementary Figure 1. Repression of hepcidin expression in the liver of mice treated with DMN Immunohistochemistry for hepcidin and H&E staining (left). qrt-pcr assays for hepcidin in the liver (right).
Supplementary Fig. 1. GPRC5A post-transcriptionally down-regulates EGFR expression. (a) Plot of the changes in steady state mrna levels versus
Supplementary Fig. 1. GPRC5A post-transcriptionally down-regulates EGFR expression. (a) Plot of the changes in steady state mrna levels versus changes in corresponding proteins between wild type and Gprc5a-/-
General Laboratory methods Plasma analysis: Gene Expression Analysis: Immunoblot analysis: Immunohistochemistry:
General Laboratory methods Plasma analysis: Plasma insulin (Mercodia, Sweden), leptin (duoset, R&D Systems Europe, Abingdon, United Kingdom), IL-6, TNFα and adiponectin levels (Quantikine kits, R&D Systems
Fig. S1. Dose-response effects of acute administration of the β3 adrenoceptor agonists CL316243, BRL37344, ICI215,001, ZD7114, ZD2079 and CGP12177 at
Fig. S1. Dose-response effects of acute administration of the β3 adrenoceptor agonists CL316243, BRL37344, ICI215,001, ZD7114, ZD2079 and CGP12177 at doses of 0.1, 0.5 and 1 mg/kg on cumulative food intake
Supplemental Data. TGF-β-mediated mir-181a expression promotes breast cancer metastasis by targeting Bim.
Supplemental Data TGF-β-mediated mir-181a expression promotes breast cancer metastasis by targeting Bim. Molly A. Taylor 1, Khalid Sossey-Alaoui 2, Cheryl L. Thompson 3, David Danielpour 4, and William
Supplementary Figure 1 Expression of Crb3 in mouse sciatic nerve: biochemical analysis (a) Schematic of Crb3 isoforms, ERLI and CLPI, indicating the
Supplementary Figure 1 Expression of Crb3 in mouse sciatic nerve: biochemical analysis (a) Schematic of Crb3 isoforms, ERLI and CLPI, indicating the location of the transmembrane (TM), FRM binding (FB)
Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex
Research article Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex Hui-Hua Li, 1 Vishram Kedar, 1 Chunlian Zhang, 1 Holly
Supplementary Figure 1
Supplementary Figure 1 Supplementary Figure 1. Neither the activation nor suppression of the MAPK pathway affects the ASK1/Vif interaction. (a, b) HEK293 cells were cotransfected with plasmids encoding
Tumour growth environment modulates Chk1 signalling pathways and sensitivity to Chk1 inhibition
Tumour growth environment modulates Chk1 signalling pathways and sensitivity to Chk1 inhibition Andrew J Massey Supplementary Information Supplementary Figure S1. Related to Fig. 1. (a) HT29 or U2OS cells
Dipeptidyl Peptidase-4 Inhibitor Increases Vascular Leakage in Retina through VE-cadherin. Phosphorylation
Dipeptidyl Peptidase-4 Inhibitor Increases Vascular Leakage in Retina through VE-cadherin Phosphorylation Choon-Soo Lee, PhD, 1,2,4 * Yun Gi Kim, MD, 3 * Hyun-Jai Cho, MD, 1,2,3 Jonghanne Park, MD, 1,2,3
Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk
Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk -/- mice were stained for expression of CD4 and CD8.
Supplementary Figure 1. EC-specific Deletion of Snail1 Does Not Affect EC Apoptosis. (a,b) Cryo-sections of WT (a) and Snail1 LOF (b) embryos at
Supplementary Figure 1. EC-specific Deletion of Snail1 Does Not Affect EC Apoptosis. (a,b) Cryo-sections of WT (a) and Snail1 LOF (b) embryos at E10.5 were double-stained for TUNEL (red) and PECAM-1 (green).
Genetic ablation of Acp1 (Lmptp) in mice prevents heart failure
Genetic ablation of Acp1 (Lmptp) in mice prevents heart failure Coralie Poizat, Ph.D. Director, Cardiovascular Research Program KFSHRC-Riyadh Saudi Heart Failure Working Group Jeddah, 5 December 2015 Cardiovascular