Supplementary Figure 1. ips_3y+mir-302b. ips_3y+mir-372. ips_3y+mir-302b + mir-372. ips_4y. ips_4y+mir-302b. ips_4y + mir-372

Transcription

1 Supplementary Figure 1 ips_3y+mir-32b ips_3y+ ips_3y+mir-32b + ips_4y ips_4y+mir-32b ips_4y + Nature Biotechnology: doi:1.138/nb.t.1862 TRA-1-6 DAPI Oct3/4

2 Supplementary Figure 1: ips cells derived from BJ cells infected with 3Y or 4Y + hsa-mir-32b and/or 372 express markers of pluripotency: Representative micrographs showing expression of TRA-1-6 and Oct3/4 in ips lines obtained by infecting BJ neonatal human foreskin fibroblasts with 3Y or 4Y+ hsa-mir-32b and/or 372. Nature Biotechnology: doi:1.138/nb.t.1862

3 Supplementary Figure 2 Expression relative to GAPDH Expression relative to GAPDH Expression relative to GAPDH Expression relative to GAPDH Expression relative to GAPDH Expression relative to GAPDH CdkN1A Rbl2 Cdc6L6 Akt1 Rab11Fip5 Rab5C Expression relative to GAPDH Expression relative to GAPDH Expression relative to GAPDH Expression relative to GAPDH Expression relative to GAPDH Y+mock 4Y+miR-32b 4Y+ 4Y+miR-32b + 3Y+mock 3Y+miR-32b 3Y+ 3Y+miR-32b + Mbd2 Smarcc2 ArhGAP26 MeCP2 TGFbR2 BJ+mock BJ+miR-32b BJ+ BJ+miR-32b+ H9 Expression relative to GAPDH Y+mock 4Y+miR-32b 4Y+ 4Y+miR-32b + 3Y+mock 3Y+miR-32b 3Y+ 3Y+miR-32b + Nature Biotechnology: doi:1.138/nb.t.1862 BJ+mock BJ+miR-32b RhoC BJ+ BJ+miR-32b+ H9

4 Supplementary Figure 2: Hsa-miR-32b and/or 372 regulate expression of a number of targets during the process of reprogramming: Graphs showing expression of individual target genes at day 7 during reprogramming after infection with either 4Y, 3Y or uninfected fibroblasts transfected with hsa-mir- 32b and/or 372. Expression was determined by qrt-pcr using samples from three independent experiments. Error bars represent standard error of mean. Nature Biotechnology: doi:1.138/nb.t.1862

5 Supplementary Figure 3 a) Number of colonies per 15, cells Mock mir-32b TβRII mir-32b + TβRII + TβRII 4Y 3Y b) TGF T RII Lenti-GFP Mock mut Lenti-T RII Mock mut p-smad3 Smad3 GAPDH c) Fold change in Venus-negative hesc-like colonies compared to mock mock mir-32b mock + TGF-β mir-32b mir TGF-β + TGF-β 4Y 3Y Nature Biotechnology: doi:1.138/nb.t.1862

6 Supplementary Figure 3: Overexpression of TβRII or TGF-β inhibits reprogramming: a) Graph showing number of colonies obtained per 15, BJ cells infected with either 4Y or 3Y. Cells were transfected with the indicated microrna mimic and/or infected with a lentivirus expressing human TβRII. N=3. Error bars represent standard deviation. b) Western blot showing levels of TβRII, phospho-smad3 and total Smad-3 in BJ cells infected with control virus (GFP) and virus expressing TβRII and treated with TGF-β for 3 mins. Cells were transfected with the indicated mirnas 48 hours prior to TGF-β treatment. c) Graph showing fold change in the number of colonies obtained from BJ cells infected with 4Y or 3Y and transfected with the indicated microrna mimic and treated with TGF-β. N=3. Error bars represent standard deviation. Nature Biotechnology: doi:1.138/nb.t.1862

7 Supplementary Figure 4 Expression relative to mock transfected sirna ctrl sirna CdkN1A Cdc2L6 ArhGAP26 Rab11Fip5 MeCP2 Rbl2 Akt1 Mbd2 Rab5C RhoC Smarcc2 Supplementary Figure 4: Validation of sirnas against targets of hsa-mir-32b and 372: Graph shows expression of individual target genes relative to mock transfected after transfection of human BJ fibroblasts with the indicated sirna pool. Error bars represent range. N=2. Nature Biotechnology: doi:1.138/nb.t.1862

8 Supplementary Figure 5 mir-32b,372 7-mer seed: mir-32b,372 mutated seed: mir-32b,372 6-mer seed: AGCACTT AGAAATT GCACTT TGFBR2 T ms1 T ms2 T ms3 T ms1,2 T ms1,3 T ms2,3 T m1,2,3 Psicheck2 TGFBR2 TmS1 TmS2 mir-32b TmS3 mir-32b,372 TmS1,2 TmS2,3 TmS3, Fold repression compared to mock Nature Biotechnology: doi:1.138/nb.t.1862 p<.5

9 Supplementary Figure 5: Luciferase analysis of TGFBR2 3 UTR seed match mutants: Seed matches for ESCC micrornas in the 3 UTRs along with different mutant constructs are shown in the top panel. Luciferase results after cotransfection with ESCC micrornas relative to mock transfection are shown in the lower panel. Ratios of Renilla luciferase readings to Firefly luciferase readings were averaged for each experiment. All data are represented as mean ± s.d. P<.5 by t-test. Nature Biotechnology: doi:1.138/nb.t.1862

10 Supplementary Figure 6 Expression normalized to GAPDH Zeb2 Expression normalized to GAPDH 1.8 Fn Expression normalized to GAPDH Ep-CAM 4Y+mock 4Y+miR-32b 4Y+ 4Y+miR-32b + 3Y+mock 3Y+miR-32b 3Y+ 3Y+miR-32b + BJ+mock BJ+miR-32b BJ+ BJ+miR-32b+ H9 p<.5 Supplementary Figure 6: Mesenchymal-to-epithelial transition occurs during the course of reprogramming: RT-qPCR showing relative expression levels of mesenchymal (Zeb2, Fn1) and epithelial (Ep-CAM) at day 7 post-infection in the process of reprogramming. RT-qPCR was performed using samples from 3 independent experiments and error bars represent standard error of mean. represents significant difference when compared to mock-transfected cells within each group (p<.5); by t-test. Nature Biotechnology: doi:1.138/nb.t.1862

11 Supplementary Figure 7 a) E-Cad β-actin 4Y+mock 4Y+ 3Y+mock 3Y+ 4Y+mock 4Y+ 3Y+mock Day 1 Day 18 3Y Y 1d 4Y + 1d 3Y 1d 3Y + 1d 4Y 18d 4Y + 18d 3Y 18d 3Y + 18d b) E-Cad β-actin 4Y+mock 4Y+ROCKi 3Y+mock 3Y+ROCKi 4Y+mock 4Y+ROCKi 3Y+mock Day 1 Day 18 3Y+ROCKi Y 1d 4Y + ROCKi 1d 3Y 1d 3Y + ROCKi 1d 4Y 18d 4Y + ROCKi 18d 3Y 18d 3Y + ROCKi 18d c) E-Cad β-actin 4Y+mock 4Y+RepSox 3Y+mock 3Y+RepSox 4Y+mock 4Y+RepSox 3Y+mock Day 1 Day 18 3Y+RepSox Y 1d 4Y + Repsox 1d 3Y 1d 3Y + Repsox 1d 4Y 18d 4Y + Repsox 18d 3Y 18d 3Y + Repsox 18d Nature Biotechnology: doi:1.138/nb.t.1862 P <.5

12 Supplementary Figure 7: ESCC micrornas, inhibition of Rho or TGF-β signaling enhance expression of E-cadherin during the course of reprogramming: Western blot showing levels of E-cadherin from lysates prepared at day 1 and 18 from BJ cells infected with either a) 4Y or 3Y +/- mir- 372, b) treated with ROCKi or c) treated with RepSox. Graphs represent ratios of E-cadherin/β-actin levels from 3 independent experiments. Error bars represent standard deviation. Nature Biotechnology: doi:1.138/nb.t.1862

13 Supplementary Figure 8 4Y 4Y+ 3Y 3Y+ d5 d8 d1 d12 d15 d18 DAPI Nature F-actin Biotechnology: doi:1.138/nb.t.1862 Scale bar = 25 um

14 Supplementary Figure 8: ESCC micrornas enhance reprogramming by regulating mesenchymal-epithelial transition: Representative micrographs showing organization of actin fibers at different days during the course of reprogramming with 4Y or 3Y +/- hsa-. Representative portions of the well are shown in each image. N=2. Scale bar represents 25µm. Nature Biotechnology: doi:1.138/nb.t.1862

15 Supplementary Figure 9 Number of colonies per 15, cells Y 4Y+miR-32b 4Y+ 4Y+miR-32b seed mutant 4Y+ seed mutant 3Y 3Y+miR-32b 3Y+ 3Y+miR-32b seed mutant 3Y+ seed mutant Day 8 Day 12 Day 14 Day 18 Supplementary Figure 9: ESCC micrornas accelerate the kinetics of reprogramming: Graph showing number of colonies at different time points during the course of reprogramming with 4Y or 3Y +/- WT or mutant ESCC micrornas. N=3. Error bars represent standard deviation. Nature Biotechnology: doi:1.138/nb.t.1862

16 Supplementary Figure 1 a) Mock mir-32b mir-32b mut mut TGF-β min TβRII TβRI p-smad2 Smad2 p-smad3 Smad3 GAPDH b) Mock mir-32b mir-32bmut mut none TGF-β c) none Scale bar = 1μm TGF-β F-actin E-cadherin DAPI F-actin E-cadherin DAPI Mock mir-32b mir-32bmut mut d) e) E-cadherin GAPDH mir-32b Nature Biotechnology: doi:1.138/nb.t.1862 mir-32bmut mut Slug mrna expression relative to RPL19 Scale bar = 2μm 8 none TGF-β Mock 32b 32bm m

17 Supplementary Figure 1: Hsa-miR-32b and 372 inhibit TGF-β-induced epithelial-mesenchymal transition in human cells: a) Western blot showing levels of TGF-β receptors, phospho-smad2, phospho- Smad3 in HaCaT cells -3min after TGF-β exposure in the presence of mirna mimics. Cells were transfected with the indicated mirnas 48 hours prior to TGFβ treatment. mir-32bmut = hsa-mir-32b seed mutant mimic, mut = hsa- seed mutant mimic. b&c) HaCaT cells were transfected with the indicated mirnas then treated or not with TGF-β for 72 h and observed by phase contrast microscopy (b), or fixed and subjected to immunostaining for F-actin, and E-cadherin (c). d) HaCaT cells were transfected with the indicated mirnas then treated with TGF-β for 72 h before lysis and immunoblotting with the indicated antibodies. e) HaCaT cells were transfected with the indicated mirnas treated or not with TGF-β for 24 h, before RNA was extracted and analyzed by RT-qPCR. Representative graph of two independent experiments is shown, Error bars represent mean ± SEM. Nature Biotechnology: doi:1.138/nb.t.1862

18 Supplementary Table 1: List of targets with the number of seed matches present in 5 UTR, open reading frame (ORF) and 3 UTR is provided. Gene Name Seed matches in 5'UTR Seed matches in ORF Seed matches in 3'UTR Total in 5'UTR AGCACTT AGCACTTA GCACTT GCACTTA Total in ORF AGCACTT AGCACTTA GCACTT GCACTTA Total in 3'UTR AGCACTT AGCACTTA GCACTT GCACTTA Total seed matches across transcript TGFBR ARHGAP ARHGEF MECP FN SMARCC RBL ZEB RAB11FIP MBD ARHGEF ZEB RBL SUV39H RHOC CCNJ LATS CDKN1A SMAD CCNG AKT INHBB ANAPC E2F RAB11FIP AKT CDC RBBP CDK2AP RALGDS RAB5C RAB11FIP TGFBRAP ARHGEF ARHGEF CHEK RBBP Nature Biotechnology: doi:1.138/nb.t.1862

19 Supplementary Table 2: List of primers used in this study AKT1 Fwd:AGCACGTGTACGAGAAGAAGCTCA Rev:ATCTTGGTCAGGTGGTGTGATGGT AKT2 Fwd:AGCACAGGTTCTTCCTCAGCATCA Rev:TGATTGTGATGGACTGGGCGGTAA ARHGAP26 Fwd:CGGCCGACCACATTTGATGTTTGA Rev: ATCTTGGCCTTCTGTTCCTCCACA ARHGEF1 Fwd: AGTCAAAGAGATCCTGCAGTGCCA Rev: AAGCCACGAAGACATCGCCTATCA ARHGEF17 Fwd:AGGGCATTGAGGGATGGAGGATTT Rev:TCTGAAAGGTCTGAGCGCAGGAAA ARHGEF18 Fwd: AAAGCCACCCGTCATCTCGTTACA Rev:GGCGCTGATCAGAAACATCGCTTT ARHGEF7 Fwd:AAGCAGCATTACAGTCCCAGACCT Rev:AGCGGACAAGAGCAAACACCTCTA CCND1 Fwd:TTGCAAGCAGGACTTTGAGGCAAG Rev:CAAACACCAGTTGGCACCAAAGGA CCNG1 Fwd:TGAGTCTGCACACGATAATGGCCT Rev:AGGTGCTTGGGCTGTACCTTCATT CCNJ Fwd: TAACCGACCCAGACTGCTTTGTGA Rev: AGTCTCCCAGGATTCATGGTGCAT CDC27 Fwd: ATGAATCCCAGGAGAGCAGCATGA Rev:ATGACACCGCCAGCTCAAGAGTAA CDC2L6 Fwd: AGCAAGCTCTGCAGGATCCCTATT Rev: CTGGTTCTGCTGCTGTTGCTGATT CDK2AP2 Fwd: ACCGCTGTTTAACGACTTTGGACC Rev:AGCATAGGTAGGCCGGATCTCTTT CDKN1A Fwd:AAGACCATGTGGACCTGTCACTGT Rev:AGAAATCTGTCATGCTGGTCTGCC CHEK2 Fwd: TGTTTCTGTTGGGACTGCTGGGTA Rev: AAGGTGGATACCCACTAAGGCAGA E2F7 Fwd: AATCCAAAGTCGTCCACACTCCCT Rev:TTGGACGACACTGTGTGACCTTGA INHBB Fwd:AGTCATTCTGTTGGGCTGTGGAGA Rev:AGTTGAGGGTTCTCGCTCTGGTTT LATS2 Fwd: AAGAGCTACTCGCCATACGCCTTT Rev:AGCTTTGGCCATTTCTTGCTCCAG MBD2 Fwd:TATCTGCTGTTGCCAGTGCTTTGC Rev:TGCGTACTTGCTGTACTCGCTCTT RAB11FIP1 Fwd:TCTGGTTTCCTCAGCAACACTCCT Rev:TTGCAAAGTGCTGGGATTACAGGC RAB11FIP5 Fwd:TGGACAGTGGTGGATGATGTGTGT Rev:TTGGAGGTGGTCAAGAGCTCGAAA RAB5C Fwd:TCTGCCTGGATGACACAACAGTCA Rev:GGCCCGTGCAAATGTATCTGTGTT RALGDS Fwd: AACAGGATCCAGCTCCCTCACAAA Rev:TGAAGGCCAGGAAGGCTGTAATGA RBBP7 Fwd:GTGACAAGGGTGAATTTGGTGGCT Rev:TGAGGATTCTGCGGCATGTAACGA RBBP9 Fwd:GTGACAAGGGTGAATTTGGTGGCT Rev: TCCAACCTATCGGCCACTTCTTGT RBL1 Fwd: AAAGGAGAATGCCTCCTGGACCTT Rev:TTAGCCTGTGCTGTCAGTTTCCCT RBL2 Fwd: AACGCTGGTTCAGGAACAGAGACT Rev:AGAAACTGGAGTCACACAAGGGCT RHOC Fwd: ACAGCAGGGCAGGAAGACTATGAT Rev:TGTCGATGGAGAAGCACATGAGGA RPL17 Fwd: ATGAGTATGCTCAGGCTACAGA Rev: GCATTGGCGATTTCATTGGTC SMAD2 Fwd: TTCAGTGCGTTGCTCAAGCATGTC Rev: AACAGTCCATAGGGACCACACACA SMARCC2 Fwd:GCTGCGCACAGACATGTACACAAA Rev:ACATTTCCAGTGCCTCCAGGAGAA SUV39H1 fwd: AGGCGAGGAGCTCACCTTTGATTA Rev: AAGAGGTATTTGCGGCAGGACTCA TGFBR2 Fwd: TGTTGAGCTCTTCAAGCAGACCGA Rev:ACTTCTCCCACTGCATTACAGCGA TGFBRAP1 Fwd: TTCTGTCTTCTGACGGACAGTGCT Rev: CGCCATTCACAATGTTCACCCACA FN1 Fwd: AAACTTGCATCTGGAGGCAAACCC Rev: AGCTCTGATCAGCATGGACCACTT ZEB1 Fwd: ATGCACAACCAAGTGCAGAAGAGC Rev: TTGCCTGGTTCAGGAGAAGATGGT ZEB2 Fwd: ATATGGTGACACACAAGCCAGGGA Rev: GTTTCTTGCAGTTTGGGCACTCGT E-CAD Fwd: TGGGCCAGGAAATCACATCCTACA Rev: TTGGCAGTGTCTCTCCAAATCCGA EP-CAM Fwd: TGTCATTTGCTCAAAGCTGGCTGC Rev: TCGCAGTCAGGATCATAAAGCCCA SLUG Fwd: TTTCTGGGCTGGCCAAACATAAGC Rev: ACACAAGGTAATGTGTGGGTCCGA OCCLUDIN Fwd: TAAATCCACGCCGGTTCCTGAAGT Rev:AGGTGTCTCAAAGTTACCACCGCT Nature Biotechnology: doi:1.138/nb.t.1862