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Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
sc-12-0446_sm_SupplFigure1.tif2348KFigure S1. GFP+ colonies induced by OKSM Representative GFP+ colonies 2 and 3 weeks after OKSM transduction of Oct4-GFPMEFs (Bar = 250 μm)
sc-12-0446_sm_SupplFigure2.tif2780KFigure S2. AP staining of induced iPSCs Alkaline Phosphatase (AP) staining of putative iPSC colonies at passage 6, with R1- ESCs as the positive controls (SC: Stat3C; Bar = 120 μm)
sc-12-0446_sm_SupplFigure3.pdf58KFigures S3 - S6. Immunostaining of putative iPSC colonies Immunostaining of putative iPSC colonies at passage 6 for Nanog (S3), SSEA-1(S4), Sox2 (S5) or Oct4 (S6), with R1-ESCs as the positive control, and R1-ESCs or iPSCs incubated with the secondary antibody only as the negative controls. Nuclei of the cells were stained with DAPI (gp: gp130Y757F; SC: Stat3C. DIC: Differential Interference Contrast; Bar = 120 μm).
sc-12-0446_sm_SupplFigure4.pdf60KFigures S3 - S6. Immunostaining of putative iPSC colonies Immunostaining of putative iPSC colonies at passage 6 for Nanog (S3), SSEA-1(S4), Sox2 (S5) or Oct4 (S6), with R1-ESCs as the positive control, and R1-ESCs or iPSCs incubated with the secondary antibody only as the negative controls. Nuclei of the cells were stained with DAPI (gp: gp130Y757F; SC: Stat3C. DIC: Differential Interference Contrast; Bar = 120 μm).
sc-12-0446_sm_SupplFigure5.pdf61KFigures S3 - S6. Immunostaining of putative iPSC colonies Immunostaining of putative iPSC colonies at passage 6 for Nanog (S3), SSEA-1(S4), Sox2 (S5) or Oct4 (S6), with R1-ESCs as the positive control, and R1-ESCs or iPSCs incubated with the secondary antibody only as the negative controls. Nuclei of the cells were stained with DAPI (gp: gp130Y757F; SC: Stat3C. DIC: Differential Interference Contrast; Bar = 120 μm).
sc-12-0446_sm_SupplFigure6.pdf66KFigures S3 - S6. Immunostaining of putative iPSC colonies Immunostaining of putative iPSC colonies at passage 6 for Nanog (S3), SSEA-1(S4), Sox2 (S5) or Oct4 (S6), with R1-ESCs as the positive control, and R1-ESCs or iPSCs incubated with the secondary antibody only as the negative controls. Nuclei of the cells were stained with DAPI (gp: gp130Y757F; SC: Stat3C. DIC: Differential Interference Contrast; Bar = 120 μm).
sc-12-0446_sm_SupplFigure7.pdf61KFigure S7. Embryoid body (EB) formation of iPSCs Original iPSCs and EB formed at days 2 and 6 of differentiation, with gradually silenced Oct4-GFP expression (4F: OKSM; 3F: OKS; SC: Stat3C; Bar = 250 μm).
sc-12-0446_sm_SupplFigure8.tif1726KFigure S8. The three germ layer differentiation of EBs Relative expression levels of the three germ layer markers and down regulation of Nanog at day 12 of EB (EB D12) differentiation (Endoderm: AFP; Mesoderm: Brachyury, Acta2; Ectoderm: Nestin, Msi1). The average gene expression values of duplicate sample preparations were normalized by GAPDH, Log10 transformed and relative to their parental iPSCs.
sc-12-0446_sm_SupplFigure9.tif1514KFigure S9. Silencing of Stat3C and Oct4 viral transgenes in iPSCs Relative expression levels of the endogenous genes (Stat3 and Oct4) and the combined retroviral and endogenous genes (*Stat3 and *Oct4) in induced iPSCs at passage 6. Silencing of the retroviral Stat3C expression was observed. The MEFs transfected with retroviral Stat3C or Oct4 were served as positive control (Ctl). The average gene expression values of duplicate sample preparations were normalized by GAPDH and relative to those in R1-ESCs.
sc-12-0446_sm_SupplFigure10.tif1460KFigure S10. G9a expression not affected by Jak/Stat3 inhibition Relative expression levels (mean±SD) of G9a and Socs3 in MEFs, R1-ESCs, and OKSM transduced MEFs treated with Jaki or DMSO at day 18 after OKSM transduction. Values were normalized by GAPDH and relative to R1-ESCs (N = 3).
sc-12-0446_sm_SupplFigure11.tif2593KFigure S11. Jak/Stat3 inhibition results in Nanog promoter hyper-methylation Upper panel: Schematic diagram of the Nanog promoter region. Arrow denotes the transcription start site. Green bar indicates the bisulfite DNA sequencing region. Lower panel: Bisulfite DNA sequencing of the Nanog promoter region for colonies isolated from OKSM reprogrammed cells with either DMSO or Jaki treatment and cultured to passage 3. MEF and R1-ESCs served as the controls. Filled and open circles represent methylated and unmethylated CpG, respectively. The percentage of total methylated CpGs for each analyzed region was shown on top of each data set.
sc-12-0446_sm_SupplFigure12.tif1480KFigure S12. Jak/Stat3 Inhibition Results in Partial Inhibition of Tet1 Expression during Reprogramming Relative expression levels (mean±SD) of Tet1/2 in MEFs treated with Jaki or DMSO 18 days after reprogramming with OKSM. Values were normalized by GAPDH and relative to R1-ESCs (N = 2).
sc-12-0446_sm_SupplFigure13.pdf57KFigure S13. Jaki blocks OKSMN reprogramming Upper: Number of GFP+ colonies (mean±SD) induced by OKSMN transduction of Oct4- GFP-MEFs reprogrammed without feeder cells, cultured in medium with either DMSO, DMSO + LIF, or 1 μM Jaki + LIF 18 days after viral transduction (**: p = 0.000, N = 2). Lower: Representative colonies from retroviral OKSMN transduced MEFs at day 18 and cultured in LIF containing reprogramming medium with either DMSO or 1 μM Jaki (Bar = 625 μm).
sc-12-0446_sm_SupplFigure14.tif2275KFigure S14. HDAC inhibition reverts arrested reprogramming by LIF deprivation Representative figures of FACS analysis of GFP+ cells at day 23 of OKSM MEF reprogramming treated with LIF antibody (LIFAb) and by VPA or TSA as described in the main text for Fig. 4c. OKSM Reprogrammed MEFs without LIF antibody and with LIF (LIF+) served as the positive control.
sc-12-0446_sm_SupplFigure15.tif1608KFigure S15. OKM-pre-iPSCs responded to Sox2 or Nanog induced reprogramming The number (mean±SD) of GFP+ colonies formed from twelve OKM-pre-iPSC lines (passage 6) previously generated by retroviral OKM transduction of MEFs. These preiPSCs were transduced with retroviral vector, Sox2, or Nanog and cultured in KSRmedium with LIF in 96-well-plates. GFP+ colonies were scored at 10 days after viral transduction under a Nikon fluorescence microscope (values represent means of 3 separate wells).
sc-12-0446_sm_SupplFigure16.tif1794KFigure S16. Jak/Stat3 activity is required in Sox2 induced OKM-pre-iPSC reprogramming GFP+ colonies (mean±SD) formed from pre-iPSC#18 in 48-well-plates (passage 6) transduced with retroviral vector, Sox2, or Sox2 plus Stat3C, cultured without LIF, and treated with either DMSO or Jaki. GFP+ colonies were scored at 14 days after viral transduction (**: p = 0.000, N = 2).
sc-12-0446_sm_SupplFigure17.tif1511KFigure S17. Inhibiting PI3K/Akt impedes but does not block OKM-pre-iPSC reprogramming GFP+ colonies (mean±SD) formed from pre-iPSC#18 in 48-well-plates (passage 6) transduced with retroviral vector, Sox2, or Sox2 plus Stat3C, cultured in KSR-medium plus LIF, with or without 10 μM LY294002. GFP+ colonies were scored at 14 days after viral transduction (**: p = 0.000, N =2)
sc-12-0446_sm_SupplFigure18.tif2891KFigure S18. RepSox induced OKM-pre-iPSC reprogramming depends on LIF signaling Colonies of OKM pre-iPSC#5 plated in 48-well-plates (passage 6) and treated with 25 μM RepSox and cultured in KSR medium with or without LIF as described in the main text for Fig. 4f (Bar = 250 μm)
sc-12-0446_sm_SupplTable1.pdf7KSupplemetary Table 1
sc-12-0446_sm_SupplTable2.pdf53KSupplemetary Table 2
sc-12-0446_sm_SupplTable3.pdf7KSupplemetary Table 3
sc-12-0446_sm_SupplTable4.pdf11KSupplemetary Table 4
sc-12-0446_sm_SupplVideo1.wmv2162KSupplementary Online Video 1: Beating cardiac cell formation from OKSM/Stat3C-induced iPSCs upon EB formation at day 12.
sc-12-0446_sm_SupplInfor.pdf89KSupplemetary Data

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