Supplemental Figure legends Figure S1. Expression of miR-133a and miR-1 in adult mice. (A) Expression of miR-133a in heart samples of miR-133a-1-KO and miR-133a-2-KO adult mice as detected by real-time PCR. Expression level of miR-133a in mutant hearts were normalized to GAPDH and compared to WT hearts. N=3 for each genotype group. (B) Northern blot analysis of heart and skeletal muscle RNA from adult mice. Ten micrograms of RNA from skeletal muscle and heart tissues were used in the Northern blots. 32P-labeled Start-Fire probes for miR-133a and miR-1 were used. Genotypes of mice are labeled on top. U6 probe was used as a loading control. GP, gastrocnemius- plantaris. (C) Expression of Mib1 mRNA and protein in dKO mice at P1. mRNA level of Mib1 as detected by real-time PCR in dKO hearts were normalized to GAPDH and compared to WT hearts. Error bars indicate SEM. Western blot analysis was performed on hearts from P1 wild type and dKO mutant mice. α -tubulin was detected as a loading control. Figure S2. Abnormal cardiomyocyte proliferation in miR-133a dKO hearts. Representative images of proliferating cardiomyocytes in WT and dKO hearts at P1. High magnification of immunohistochemistry on heart sections at P1 using phospho- histone H3 (PH3, red) and -actinin (green) was shown. Bar = 20 m. Figure S3. Expression of miR-133a in wild-type and beta-MHC-miR-133a transgenic hearts at E13.5 as detected by real-time PCR. Figure S4. Expression of miR-133a target genes in hearts of WT and dKO mutant mice at P1. (A) mRNA levels as detected by real-time PCR for each gene in dKO hearts were normalized to GAPDH and compared to WT hearts. Error bars indicate SEM. Whsc2: Wolf-Hirschhorn syndrome candidate 2; Rhoa: Ras homolog gene family, member A; PTBP2: polypyrimidine tract binding protein 2; HERG: human Ether-a-go-go related Gene. (B) Protein expression of caspase 9 and Rhoa in WT and dKO mutant hearts. Western blot analysis was performed on hearts from P1 wild type (n = 3) and dKO mutant (n = 3) mice. α -tubulin was detected as a loading control. Quantification of bands by densitometry showed no significant increase in expression of caspase 9 and Rhoa in dKO compared to WT hearts. Supplemental Tables Supplemental Table1: Summary of mating to obtain miR-133a-1KO/KO mice and miR-133a-2KO/KO mice Breeding Total mice KO/KO obtained KO/+ obtained +/+ obtained (expected) (expected) (expected) miR-133a1 KO 111 25 (28) 59 (55) 28 (28) miR-133a2 KO 128 26 (32) 61 (64) 41 (32) 1 Supplemental Table 2. Upregulated genes in miR-133a dKO hearts. Category Genes dysregulated in miR-133a dKO hearts Fold- Predicted increase Targets Symbol Description cytoskeletal KRT18 keratin 18 3.8 protein KIF22 kinesin family member 22 3.3 TPX2 microtubule-associated protein homolog 3.0 KIF2C kinesin family member 2C 2.8 DNAJB6 DnaJ (Hsp40) homolog, subfamily B, member 6 2.6 ANLN anillin, actin binding protein 2.5 MYH9 myosin heavy chain 9, non muscle 2.5 Y Csrp cysteine and glycine-rich protein 1 2.4 SLC4A4 solute carrier family 4, member 1 2.4 Csrp2 cysteine and glycine-rich protein 2 2.1 PDLIM5 PDZ and LIM domain 5 2.1 ACTN1 actinin, alpha 1 2.0 CNN3 calponin 3 2.0 PFN2 profilin 2 1.5 Y Sarcomeric TPM1 tropomyosin 1, alpha 5.6 proteins Acta2 smooth muscle alpha-actin 4.6 TAGLN transgelin 4.1 CALD caldesmon 1 3.4 CNN1 calponin 1 3.4 TMP2 tropomyosin 2, beta 2.3 MYH7 myosin heavy chain 7 2.1 TAGLN2 transgelin 2 2.0 Y CNN2 calponin 2 2.0 Y TNN1 Troponin I type 1 slow skeletal 1.5 Cell cycle/ RBBP9 retinoblastoma binding protein 9 4.0 apoptosis PPP2R2A protein phosphatase 2, regulatory subunit A, alpha isoform 3.9 CENPE centromere protein E 3.0 DDX3Y DEAD box polypeptide 2, Y-linked 3.0 Y CDC20 cell division cycle 20 homolog 2.8 CDCA8 cell division cycle associated 8 2.7 CDCA1 cell division cycle associated 1 2.7 TTK Ttk protein kinase 2.7 BCL2L2 Bcl2-like 2 2.4 Y CKS2 CDC28 protein kinase regulatory subunit 2 2.4 BCL2L1 Bcl2-like 1 2.3 Y CCNB1 cyclin B1 2.3 CLU clusterin 2.1 CCND1 cyclin D1 2.1 2 ECT2 epithelial cell transforming sequence 2 oncogene 2 LASS2 longevity assurance homolog 2 1.9 Y CCND2 cyclin D2 1.5 Y CCNC cyclin C 1.5 Y Signaling TRO trophinin 5.6 THBS4 thrombospondin 4 4.7 PBK PDZ binding kinase 3.7 ARHGAP11A Rho GTPase activating protein 11A 2.4 GPR126 G protein-coupled receptor 126 2.4 RABL2A RAB, member of RAS oncogene family-like 2A 2.4 SDC4 syndecan 4 2.4 BMP2 bone morphogenetic protein 2 2.3 BMP6 bone morphogenetic protein 6 2.3 NPPB natriuretic peptide precursor B 2.2 Rasl11b RAS-like, family 11, member B 2.2 TGFB1 transforming growth factor, beta induced 2.2 Racgap1 Rac GTPase-activating protein 1 2.1 Itga5 integrin alpha 5 (fibronectin receptor alpha) 2.0 SRF serum response factor 1.5 Y Supplemental Table 2. Up-regulated genes in miR-133a dKO mutant hearts at P1. This list comprises the up-regulated genes in the heart of miR-133a dKO mutant mice at P1 by microarray analysis (cut-off 1.5-fold). The gene categories listed include up- regulated genes that are over-represented in the array shown by PANTHER microarray analysis tools. Fold increase is shown. Y indicates that the gene is a predicted miR-133a target. 3 110221_Liu_SuppFig1 A 0.8 0.6 0.4 Relative 0.2 miR-133a Level 0 a1KO a2KO B dKO a2-KO a1-KO;a2-het a1-het;a2-het dKO a2-KO a1-KO;a2-het a1-het;a2-het WT a1-KO a1-het;a2-KO WT a1-KO a1-het;a2-KO miR-133a miR-1 U6 Adult Skeletal Adult Heart Muscle (GP) C 1.5 WT dKO 1.0 Mib1 Level α-Tubulin Relative 0.5 mRNA 0 WT dKO 110221_Liu_SuppFig2 P-H3 α-actinin WT dKO 110221_Liu_SuppFig3 20 15 10 5 0 Relative miR-133a level WT Tg 110221_Liu_SuppFig4 A 4 WT Level 3 dKO 2 1 Relative mRNA 0 cdc42 whsc2 Rhoa PTBP2 caspase 9 HERG B WT dKO WT dKO Caspase 9 Rhoa α-tubulin α-tubulin 0.8 1.5 0.6 1.0 0.4 0.5 0.2 Rhoa / tubulin caspase 9 / tubulin 0 0 WT dKO WT dKO.