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sc-12-0890_sm_SupplFigure1.pdf209KFigure S1 Knockdown of Mbd3 increases the efficiency of iPSC generation. (A) Western blot showing the expression of Mi-2, HDAC1&2 and Mbd2&3 in MEFs, iPSCs and mESCs. (B-D) MEFs infected with Oct4/Sox2/Klf4/c-Myc (OSKM) in the presence of different Mbd3-specific RNAs were cultured in mESC medium and assayed for reprogramming 16 days after infection. (B) Left: Average numbers of AP+ and Oct4-GFP+ clones from 1.5×104 MEFs. Data are from three independent experiments; error bars represent standard deviation. Right: FACS analyses showing the percentage of Oct4-GFP positive cells. (C) Representative experiments showing differential interference contrast (DIC) and fluorescent images of MEFs, scale bars = 300 μm. (D) RT-PCR analysis of mESC marker genes. (E-F) Characterization of iPSCs derived from MEFs expressing OSKM and shMbd3. (E) AP staining and immunofluorescence of mES marker genes (SSEA-1 and Nanog). (F) Immunofluorescence showing embryoid body (EB)-mediated differentiation of iPSCs into ectoderm, mesoderm and endoderm using antibodies against Nestin, SM-actin and ARF. Scale bars = 100 μm. Abbreviations: MEF, mouse embryonic fibroblast; iPSCs, induced pluripotent stem cells; mESCs, mouse embryonic stem cells; RT-qPCR, reverse transcription quantitative polymerase chain reaction; OSKM, Oct4, Sox2, Klf4, c-Myc; DIC, differential interference contrast; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorting; AP, alkaline phosphatase; SM-Actin, smooth muscle actin; AFP, α-fetoprotein.
sc-12-0890_sm_SupplFigure2.pdf74KFigure S2 Depletion of Mbd3 does not alter cell cycle progression. (A) Cell proliferation assay. MEF cells were infected with retroviruses encoding control (shCtrl) or Mbd3-specific shRNAs (shMbd3-1, 2&3). Cell numbers were counted in 24 h intervals 5-11 days after infection. (B) MEFs were infected with retroviruses encoding control (shCtrl) or Mbd3-specific shRNAs and subjected to FACS analysis 10 days after infection. Abbreviations: MEF, mouse embryonic fibroblast; FACS, fluorescence-activated cell sorting.
sc-12-0890_sm_SupplFigure3.pdf125KFigure S3 Depletion of Mbd3/NuRD substitutes for Sox2 in iSF1 medium. (A) Strategy for iPSC induction in iSF1 culture medium. (B) Knockdown of Mbd3 replaces Sox2 in iSF1 medium. Top: 2,500 MEFs infected with Oct4/Sox2/Klf4/c-Myc (OSKM) alone or together with Mbd3 or shMbd3 were cultured in iSF1 medium and assayed for reprogramming 12 days after infection. AP- and Oct4-GFP-positive clones were counted (top left) and the percentage of Oct4-GFP positive cells was analyzed FACS (top right). Bottom: 1.5×104 MEFs infected with Oct4/Sox2/Klf4 (OSK) alone or together with Mbd3 or Mbd3 shRNA were cultured in iSF1 medium and assayed for reprogramming 18 days after infection. AP- and Oct4-GFP-positive clones were counted (bottom left) and the percentage of Oct4-GFP-positive cells was monitored by FACS (bottom right). Error bars represent standard deviation. (C) RT-PCR analysis of showing expression of mESC marker genes in Oct4/ Klf4/c-Myc (OKM) plus shMbd3 derived iPS cells (sriPS). (D) 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) based assay monitoring viability and proliferation of sriPS cells. Abbreviations: MEFs, mouse embryonic fibroblasts; iPSC, induced pluripotent stem cell; mESC, mouse embryonic stem cell; RT-PCR, reverse transcription polymerase chain reaction; OSKM, Oct4, Sox2, Klf4 and c-Myc; OSK, Oct4, Sox2 and Klf4; OKM, Oct4, Klf4 and c-Myc; GFP, green fluorescent protein; AP, alkaline phosphatase; FACS, fluorescence-activated cell sorting; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide.
sc-12-0890_sm_SupplFigure4.pdf140KFigure S4 Microarray analysis of gene expression in control and Mbd3-depleted MEFs. (A) Cluster analysis of Mbd3 regulated genes in mES cells, Mbd3-depleted and VPA treated MEFs. Individual genes are listed in Table S2. (B) Immunoblots showing the expression of iPSC promoting factors upon depletion of Mbd3. MEFs were infected with retroviruses encoding control (shCtrl) or Mbd3-specific shRNAs (shMbd3-1&2) and the level of Nanog, Oct4 and Sox2 was monitored 10 days after infection. (C) Cluster analysis of Mbd3 target genes that are involved in activation of MAPK. Left: Gene expression in mESC, Mbd3-depleted and VPA-treated MEFs. Right: Binding of Mbd3 or Mbd3-containing complexes to gene promoters Individual genes are listed in Table S3. Abbreviations: VPA, valproic acid; MEF, mouse embryonic fibroblast; iPSC, induced pluripotent stem cell; mESC, mouse embryonic stem cell; MAPK, mitogen-activated protein kinase.
sc-12-0890_sm_SupplFigure5.pdf94KFigure S5 Gene Ontology clustering of Mbd3 regulated genes. Gene Ontology analysis of Mbd3 regulated genes based on the GO categories (A) biological process and (B) molecular function.
sc-12-0890_sm_SupplInfor.pdf8KSupplemental Data
sc-12-0890_sm_SupplTable1.pdf15KSupplemental Table 1
sc-12-0890_sm_SupplTable2.pdf2350KSupplemental Table 2
sc-12-0890_sm_SupplTable3.pdf53KSupplemental Table 3

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