Supporting Information Zhou et al. 10.1073/pnas.1516237112 SI Materials and Methods to calculate fold change and P values. Cutoff values of fold Differential Gene Expression Analysis. Three micrograms of DNase- change greater than two and P value less than 0.01 were then treated RNA per replicate were prepared using the TruSeq used to select for differentially expressed genes between sample Stranded LT Kit from Illumina. Samples were then PCR am- group comparisons. plified and purified with Ampure XP beads before sequencing on a HiSeq 2500 instrument (Illumina). All procedures were carried Pathway Enrichment Analysis. Differentially expressed genes from out according to manufacturer protocols. Quality assessment of the RNA expression data are associated with a biological function the RNA-Seq data was done using NGS-QC Toolkit. Reads supported by at least one publication in the Ingenuity Pathways with mean phred quality scores of less than 20 were removed from Knowledge Base. Fisher’s exact test was then used to calculate further analysis. Quality filtered reads were then aligned to the the P value and determine the probability that each biological mouse reference genome GRCm38 (mm 10) using the Bowtie2 function was enriched in the dataset due to chance alone. Sta- (v 2.0.6) aligner (18). Only uniquely mapped reads were kept for tistically significant biological pathways were then identified by further downstream analysis. Differential gene expression anal- selection for pathways with P values less than 0.05. The DAVID ysis was done using the R package DESeq (v 1.10.1) (19) following gene functional annotation and classification tool (21) was used the protocols outlined in ref. 20. Read counts were normalized by to annotate the list of differentially expressed genes with re- taking the median of each gene count across samples and dividing spective Gene Ontology (GO) terms and perform GO enrich- each sample gene count by the relative ratio of library sizes be- ment analysis for molecular and biological functional categories. tween the calculated median and sample size. The averaged Functional Gene Ontology groups were selected for significance normalized expression values of the triplicate samples were used by using a P value cutoff of 1%. Zhou et al. www.pnas.org/cgi/content/short/1516237112 1of8 Fig. S1. Akt enhances cardiac reprogramming by GHMT. (A) Western blot of Akt and phospho-Akt in TTFs 7 d after infection with the indicated retroviral Akt + + expression cassettes. (B–D) Representative flow cytometry plot and (E–G) analyses of αMHC-GFP and cTnT cells in MEFs, CFs, or TTFs, after infection with control, Akt, GHMT, or AGHMT retrovirus at indicated times (5 d for MEFs, 1 wk for CFs and TTFs). #P < 0.05 vs. all others. Zhou et al. www.pnas.org/cgi/content/short/1516237112 2of8 Fig. S2. Strategy for measuring calcium flux. MEFs derived from αMHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 transgenic mice were reprogrammed to iCMs by addition of GHMT or AGHMT and exhibit spontaneous cyclic autofluorescence concomitant with calcium flux. Fig. S3. Addition of Akt1 to GHMT stimulates maturation of iCMs in TTFs. (A) α-Actinin immunostaining shows binucleate iCM 3 wk after induction with GHMT and AGHMT. (Scale bars: 25 μm.) (B and C) Quantification of the percentage of iCMs with more than a single nucleus (binucleate, two nuclei; multi- nucleate, three or more nuclei) 3 wk after AGHMT treatment reveals an increase relative to cells treated with GHMT. (D) Size of iCMs was approximately doubled by addition of Akt1 to GHMT, as shown here 3 wk after induction after immunocytochemistry for α-actinin. (E) The ratio of Myh6:Myh7 by qPCR in TTFs 1 wk after induction suggests that adding Akt1 to GHMT results in more mature iCMs whereas a kinase dead mutant of Akt1 abrogates this effect. #P < 0.05 vs. all others. Zhou et al. www.pnas.org/cgi/content/short/1516237112 3of8 Fig. S4. RNA-Seq global analysis shows that AGHMT iCMs are more similar to mature CM than GHMT iCMs. (A) Multidimensional scaling (MDS) plot showing RNA-Seq sample relatedness based on 2D coordinates. Distance measurements were calculated using normalized RNA expression values from all expressed markers in each sample. (B) Count table for differentially expressed markers for various sample group comparisons using fold change cutoff of ≥2 and P value of ≤0.01. (C) Venn diagram showing number of overlapping markers between GHMT, AGHMT, and CM compared with empty vector control. Marker counts include both up-regulated and down-regulated genes. Zhou et al. www.pnas.org/cgi/content/short/1516237112 4of8 Fig. S5. Processes not involved in the mechanism by which Akt1 enhances GHMT-mediated formation of iCMs. (A) Measurement of Gata4, Hand2, Mef2c, or Tbx5 transcript by qPCR after 7 d of induction in GHMT- or AGHMT-treated MEFs. (B ) Western blot of myc-tagged GHMT proteins in the presence or absence of Akt1 in MEFs after 2 d of induction. (C) Flow cytometry analysis after 7 d of GHMT treatment in MEFs (derived from aMHC-GFP mice) in the presence or absence of CHIR99021 (CHIR) or BIO. (D and E ) Flow cytometry analysis of MEFs treated with Edu for 1 h following 7 d induction with control, Akt, GHMT, or AGHMT. Cardiac troponin T (cTnT) labels iCMs and Edu labels proliferating cells. (F) Flow cytometry analysis was used to measure apoptosis of MEFs following 7 d in- duction with control, Akt, GHMT, or AGHMT. Addition of Akt had no significant effect on apoptosis as measured by this assay. *P < 0.05; #P < 0.05 vs. all others; &P < 0.05 vs. all others unless also labeled with “&”. Zhou et al. www.pnas.org/cgi/content/short/1516237112 5of8 Table S1. Summary of RNA-Seq sequencing output, quality control filtering, and mapping results Sample Total reads Filtered % Mapped Mapping ratio, % Unique reads Unique ratio, % GHMT-1 7,276,236 7,056,944 97.0 5,813,947 82.4 2,462,467 42.4 GHMT-2 6,377,141 5,815,703 91.2 3,835,725 66.0 2,745,984 71.6 GHMT-3 5,392,897 4,945,779 91.7 3,473,624 70.2 2,258,765 65.0 AGHMT-1 31,744,595 31,526,448 99.3 26,701,703 84.7 16,869,653 63.2 AGHMT-2 33,918,839 33,689,150 99.3 28,545,217 84.7 19,429,545 68.1 AGHMT-3 41,491,341 41,213,099 99.3 34,529,127 83.8 22,427,626 65.0 CM-1 40,783,401 40,584,054 99.5 34,931,197 86.1 17,989,722 51.5 CM-2 35,168,486 34,997,535 99.5 29,953,628 85.6 15,163,436 50.6 CM-3 10,713,123 10,650,974 99.4 9,109,740 85.5 5,716,551 62.7 E-1 57,253,655 56,875,215 99.3 48,706,296 85.6 40,326,320 82.8 E-2 29,011,588 28,455,694 98.1 23,808,068 83.7 19,260,544 80.9 E-3 32,718,600 32,524,244 99.4 27,952,054 85.9 22,782,625 81.5 Table S2. List of myristoylated kinases tested in an addition analysis to enhance GHMT-mediated cardiac reprogramming AAK1 CKMT1A HK1 MVK PIP5K1B RPS6KL1 ACVR1 CKMT2 HK2 NADK PIP5K2A RPSK6A3 ADCK4 CKS1B HK3 NEK11 PIP5K3 SGK ADCK5 CKS2 IHPK2 NEK3 PKM2 SNF1LK ADPGK CLK1 IKBKE NEK6 PKN1 SPHK2 ADRBK1 CLK2 ILK NME7 PKN2 SRPK2 ADRBK2 CLK3 ITK NTRK3 PLAU STK17B AKT1 CMPK ITPK1 NUAK2 PLK1 STK3 AKT3 CSNK1A1L ITPKB OXSR1 PLK2 STK32A AMHR2 CSNK1E LCK PACSIN1 PLK3 STK32B AURKA CSNK1G1 LIMK1 PAK4 PLK4 STK32C AXL CSNK1G2 LIMK2 PAPSS1 PMVK STK33 BLK DAK MAP2K5 PBK PNKP STK38L BMX DGKG MAP2K6 PCK2 PRKAA1 STK4 BTK DGUOK MAP2K7 PCTK1 PRKACB STK40 CALM2 DLG5 MAP3K14 PCTK2 PRKACG SYK CAMK1G DYRK2 MAP3K6 PCTK3 PRKAG2 TAOK3 CAMK2B DYRK4 MAP3K7 PDIK1L PRKAR2A TBK1 CAMK2D EPHA4 MAP3K8 PDK1 PRKCD TEC CAMK4 FASTK MAPK12 PDPK1 PRKCI TESK1 CAMKK1 FGFR1 MAPK13 PDXK PRKCZ TIE1 CAMKV FGR MAPK14 PFKL PRKRA TK1 CDC2 FRK MAPK6 PFKM PTK2 TNK2 CDK2 GAK MAPK7 PI4K2B RET TSSK1B CDK4 GALK2 MAPKAP1 PIK3CB RIOK1 TSSK6 CDK5 GCK MAST1 PIK3CG RIOK2 TTK CDK7 GK MATK PIK3R3 RIOK3 TYK2 CDK9 GK2 MELK PIK3R5 RPS6KA2 UCK2 CERK GRK5 MKNK1 PIK4CA RPS6KA5 ULK4 CHEK1 GRK6 MOBKL1A PIK4CB RPS6KA6 VRK2 CKB HCK MOBKL2A PIM1 RPS6KB1 VRK3 CKM HIPK1 MPP1 PIP5K1A RPS6KB2 YES1 Zhou et al. www.pnas.org/cgi/content/short/1516237112 6of8 Movie S1. Spontaneous calcium flux in reprogrammed (by GHMT or AGHMT) mouse embryonic fibroblasts (derived from Rosa26A-GCamp3+/αMHC-Cre+ mice). Movie S1 Movie S2. Contraction time course of AGHMT- and GHMT-treated reprogrammed mouse embryonic fibroblasts. Movie S2 Zhou et al. www.pnas.org/cgi/content/short/1516237112 7of8 Movie S3. Contraction of AGHMT-treated reprogrammed cardiac fibroblasts at 2 wk and tail tip fibroblasts at 3 wk after induction. Movie S3 Movie S4. Contraction of AGHMT-treated reprogrammed mouse embryonic fibroblasts 3 wk after induction at baseline and in response to β-adrenoreceptor pharmacologic manipulation. Movie S4 Zhou et al. www.pnas.org/cgi/content/short/1516237112 8of8.