Supplemental Information. A Mesenchymal-to-Epithelial Transition. Initiates and Is Required for the Nuclear. Reprogramming of Mouse Fibroblasts

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1 Cell Stem Cell, Volume 7 Supplemental Information A Mesenchymal-to-Epithelial Transition Initiates and Is Required for the Nuclear Reprogramming of Mouse Fibroblasts Ronghui Li, Jialiang Liang, Su Ni, Ting Zhou, Xiaobing Qing, Huapeng Li, Wenzhi He, Jiekai Chen, Feng Li, Qiang Zhuang, Baoming Qin, Jianyong Xu, Wen Li, Jiayin Yang, Yi Gan, Dajiang Qin, Shipeng Feng, Hong Song, Dongshan Yang, Biliang Zhang, Lingwen Zeng, Liangxue Lai, Miguel Angel Esteban, and Duanqing Pei 1

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3 Figure S1-related to Figure 1 a, Schematic representation of early EMT processes during development and MET during somatic cell reprogramming. b, Immunofluorescence microscopy for the indicated markers in MECs, MEFs, ipscs, and R1 ESCs. c, qpcr for the indicated cell-matrix adhesion related genes in SKOM infected MEFs compared to control, mean values + SD of 3 independent time course experiments are shown. d, qpcr for the indicated micrornas in MECs, R1 ESCs, and 2 ipsc clones; values are referred to MEFs. e, Quantification of cell migration through Matrigel coated- Transwell filters after 36 hours. f, Quantification using ImagePro 5.0 of gap closure after scratching cell monolayers with a tip edge; the monolayers were washed and medium changed after the scratching. 3

4 Figure S2-related to Figure 2 Measurement of proliferation (by counting cells in triplicate) in MEFs transduced with SKOM (at day 6) or SKO factors (at day 9) and treated with Tgfb cytokines or co-expressing Snail. N=6 for SKOM + Tgfb1; N=3 for SKOM + Tgfb2, N=3 for SKOM + Tgfb3, N=9 for SKOM + Snail; N=4 for SKO + Tgfb1, N=4 for SKO + Tgfb3, N=4 for SKO + Snail). 4

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6 Figure S3-related to Figure 3 a, qpcr for the indicated genes in 2 ipsc clones compared to MEFs. b, Scatter plot representation of DNA microarrays comparing the gene expression profiles of R1 ESCs, MEFs, and an ipsc cell line. Specific genes are highlighted and the expression difference is shown. c, Immunofluorescence microscopy of R1 ESCs and the same ipscs cultured in mouse ESC medium + added Tgfb1 for 3 passages (3-5 days in culture each). d, qpcr for the indicated genes of 5 different stably GFP- pre-ipsc clones generated from MEFs using SKOM factors. Values are referred to non transduced MEFs, R1 ESCs and ipscs were included as controls. e, Measurement of proliferation (by counting cells in triplicate) at days 6 (SKOM) and 9 (SKO) in MEFs transduced with SKO and treated with Alk5i or co-expressing Smad7. N=9 for SKOM + Alk5i, N=3 for SKOM + Smad7, N=9 for SKO + Alk5i, N=3 for SKO + Smad7). 6

7 Figure S4-related to Figure 5 a, Hierarchical clustering of the gene expression profiles of the 3 control and 3 shecad cell lines; shecad 4 and 8 are more similar between themselves than with shecad 9. b, Number of genes (DE list) differentially expressed in shecad cell lines (average of the 3) compared to the control (average of the 3), a 1.5 fold cut off value was used. c, Schematic representation showing the number of genes differentially expressed or shared (1.5 fold cut off) between the 3 individual shecad cell lines compared to the average of the 3 controls. d, Heat map of selected genes in the DE list that belong to Gene Ontology (GO) categories related to sexual reproduction. 7

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9 Figure S5-related to Figure 6 Additional characterization of ipsc clones produced from MECs as in Figure 6 b. a, Phase contrast and GFP fluorescence photographs, and immunofluorescence for the indicated markers. b, Upper panel: qpcr for the indicated ESC genes, R1 ESCs were used as positive control. Lower panel: qpcr for the transgenes, transduced cells extracted at day 6 and uninfected MECs were the positive and negative controls respectively. c, DNA methylation profile of the Nanog proximal promoter. d, Normal karyotype. e, Teratoma sections. f, Chimeric mouse. 9

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11 Table S1-related to Figure 2 DNA microarrays of SKOM-transduced MEFs with added Tgfb1 or co-expressing Snail and analyzed at day 10, differentially expressed genes and selected KEGG pathways related to EMT (analyzed with the David Bioinformatics Database [ are shown. Table S2 (provided separately as an Excel file available online)-related to Figure 5 a, Distribution of GO categories related to sexual reproduction for genes in the DE list (as in Supplementary Figure 4). b, List of these same genes (as in a) and their corresponding GO categories. c, Selected KEGG pathways (analyzed with DAVID) of genes in the DE list. Specific genes belonging to the Tgfb pathway are highlighted. E-cadherin is also highlighted and the fold expression values for the 3 individual shecad cell lines relative to the average of the 3 controls are shown. 11