SUPPLEMENTARY INFORMATION

Transcription

1 Supplementary Figure a 3 factors 4 factors Nanog-GFP phase b c.5 Endogenous Oct3/4 2 Total Oct3/4.5 Relative expression (ES = ) Endogenous Sox2 Nanog Relative expression (ES = ) Total Sox2 Total Klf p53 -/- p53 +/- MEF MEF ES 6 Total c-myc 4 factors 3 factors p53 -/- p53 +/- MEF MEF ES 4 factors 3 factors

2 d 3 factors 4 factors e 3 factors 4 factors Nanog-GFP phase Supplementary Figure Characterization of ips cells derived from p53 heterozygous or homozygous MEFs a. Shown are phase contrast images (upper) and fluorescent images (lower) of ips cells (passage 2) derived from Nanog-GFP, p53-null MEFs by three or four (+ c-myc) reprogramming factors. Bars indicate μm. b. The expressions of Nanog, endogenous Oct3/4 and endogenous Sox2 were analyzed by real-time PCR. Shown are relative expression levels compared to those in ES cells. c. The expressions of the four factors were analyzed by real-time PCR. Primer sets in these experiments amplify both the endogenous genes and transgenes. Shown are relative expression levels compared to those in ES cells. d. Histological examination of teratomas derived from p53-null ips cells with the three or four factors. Shown are hematoxylin-eosin staining of neural tissues (upper left), cartilage (upper right), muscle (lower left), and fat tissues (lower right) for three factor-ips cells, while undifferentiated cells for four factor-ips cells. e. Shown are phase contrast images (upper) and fluorescent images (lower) of the same clones in a at passage 5. Bars indicate μm. 2

3 Supplementary Figure 2 a 4 factors b 3 factors c 5 2 * 6 p53 4f + Mock 4f + p53 shrna- 4f + p53 shrna-2 4f + p53 shrna-2 + mp53 4f + RB shrna + DsRed + p53 P2 + p53 DD + p53 W + p53 WT + p53 DD + p53 P275S + DsRed RB β-actin 75S T d factors e factors + RB shrna + p53 shrna-2 + p53 shrna- + Mock + p53 shrna-2 + p53 shrna-2 + DsR + RB shrna + p53 shrna-2 + p53 shrna- + Mock + mp53 + p53 shrna-2 + mp53 + p53 shrna-2 + DsRed ed 3

4 f Supplementary Figure 2 Increased efficiency of human ips cell generation by p53 suppression a. The retroviral vector expressing either p53 P275S or p53dd was transduced into HDFs together with the four reprogramming factors. Shown are the numbers of ips cell colonies from 5 x 3 HDFs. *; p<.5 compared to DsRed control (n=3). b. The retroviral vector expressing either p53 P275S or p53dd was transduced into HDFs together with the three reprogramming factors. Shown are the numbers of ips cell colonies from 4 x 4 HDFs. ; p<. compared to DsRed control (n=3). c. Retroviral vectors for p53 shrna, RB shrna, or control vector were transduced into HDFs. Six days after the transduction, protein levels of p53, RB, and β-actin were determined by western blot analyses. d. The retroviral vector expressing p53 shrna, RB shrna, or control vector was transduced into HDFs together with the four reprogramming factors. To rescue RNAi-mediated knockdown, a retroviral vector for mouse p53 was co-introduced. Shown are the numbers of ips colonies from 5 x 4 cells of HDF. ; p<. compared to DsRed control (n=4). e. The retroviral vector expressing p53 shrna, RB shrna, or control vector was transduced into HDFs together with the three reprogramming factors. Shown are the numbers of ips colonies from 5 x 4 cells of HDF. ; p<. compared to DsRed control (n=4). f. Teratomas derived from human ips cells, which were generated with the three reprogramming factors and the p53 DD mutant. Shown are hematoxylin-eosin staining of neural tissues (upper left), cartilage (upper right), muscle (lower left), and gut-like epithelial tissues (lower right). 4

5 a seed X 5 cells infection with DsRed reseed 5 cells count DsRed positive spots - day 5 b No. of DsRed + spots Supplementary Figure 3 Effect of p53 deletion on retroviral transduction and plating efficiency a. MEFs (p53 +/- or p53 -/- ) were transfected with a retrovirus expressing DsRed and reseeded (5 cells/ mm-dish) next day. b. Numbers of DsRed-positive colonies were counted on day 5. 5

6 Supplementary Table Common gene lists regulated by p53 in mouse and human Increased genes Accession No. Gene Name NM_89 Homo sapiens gamma-aminobutyric acid (GABA) A receptor, alpha 4 (GABRA4) NM_82767 Homo sapiens solute carrier family 6 (neutral amino acid transporter), member 5 (SLC6A5), transcript variant NM_4264 Homo sapiens polo-like kinase 4 (Drosophila) (PLK4) NM_9839 Homo sapiens fibronectin leucine rich transmembrane protein 3 (FLRT3), transcript variant 2 NM_7998 Homo sapiens RAB39B, member RAS oncogene family (RAB39B) NM_482 Homo sapiens glycine amidinotransferase (L-arginine:glycine amidinotransferase) (GATM), nuclear gene encoding mitochondrial protein NM_373 Homo sapiens v-myb myeloblastosis viral oncogene homolog (avian) (MYB), transcript variant NM_44 Homo sapiens gap junction protein, beta 2, 26kDa (GJB2) Decreased genes Accession No. Gene Name NM_546 Homo sapiens tumor protein p53 (TP53), transcript variant NM_389 Homo sapiens cyclin-dependent kinase inhibitor A (p2, Cip) (CDKNA) NM_46 Homo sapiens flavin containing monooxygenase 5 (FMO5) NM_2474 Homo sapiens myosin, heavy chain, smooth muscle (MYH), transcript variant SMA NM_6763 Homo sapiens BTG family, member 2 (BTG2) NM_4455 Homo sapiens exostoses (multiple)-like (EXTL) NM_2247 Homo sapiens zinc finger, matrin type 3 (ZMAT3), transcript variant NM_6536 Homo sapiens CLCA family member 2, chloride channel regulator (CLCA2) NM_795 Homo sapiens inositol polyphosphate-5-phosphatase, 45kDa (INPP5D), transcript variant NM_6879 Homo sapiens Mdm2, transformed 3T3 cell double minute 2, p53 binding protein (mouse) (MDM2), transcript variant MDM2b NM_4469 Homo sapiens c-fos induced growth factor (vascular endothelial growth factor D) (FIGF) NM_6352 Homo sapiens carboxypeptidase A4 (CPA4) NM_2487 Homo sapiens thrombospondin, type I, domain containing 4 (THSD4) NM_526 Homo sapiens keratin 4 (KRT4) NM_846 Homo sapiens glutathione S-transferase A2 (GSTA2) NM_98 Homo sapiens hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2 (HSD3B2) NM_32866 Homo sapiens cingulin-like (CGNL) NM_23 Homo sapiens leptin (LEP) NM_245 Homo sapiens plexin domain containing (PLXDC) NM_4975 Homo sapiens potassium voltage-gated channel, Shab-related subfamily, member (KCNB) NM_283 Homo sapiens SPARC-like (hevin) (SPARCL), transcript variant NM_32588 Homo sapiens tripartite motif-containing 63 (TRIM63) NM_2247 Homo sapiens differentially expressed in FDCP 6 homolog (mouse) (DEF6) NM_3322 Homo sapiens tubby like protein (TULP) NM_32 Homo sapiens secreted frizzled-related protein (SFRP) NM_264 Homo sapiens indoleamine-pyrrole 2,3 dioxygenase (INDO) NM_46 Homo sapiens cyclin G (CCNG), transcript variant 6