Figure S1. Transformed human epithelial cells showed up-regulated p63 but down-regulated p53. (a) Heavy particle radiation promotes human bronchial

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1 Figure S1. Transformed human epithelial cells showed up-regulated p63 but down-regulated p53. (a) Heavy particle radiation promotes human bronchial cell transformation. The immortalized cells without IR (LE1) or exposed to Fe (1 GeV/u) either 0.2 Gy x 5 in 5 days (LET1-1) or 0.5 Gy (LET1-2) were subcultured for 4 months. The cell transformation was detected by growing colonies in soft-agar. The non-irradiated LE1 cells did not grow any colonies in this condition. Data shown are the mean + SD from three independent experiments. (b) The levels of p63 measured using Western blot from whole cell lysates of human LE1 (#1), LET1-1 (#2) and LET1-2 cells (#3). Lane 1 (M) is the marker with different molecular weights. PCNA was used as an internal loading control. (c) Upper panel: the levels of p53 or p63 in human transformed epithelial cells T and their non-transformed control cells C. As described in Supplementary Table 1: 1. LE1, 2. LET1-1, 3. LET1-2, 4. LE2, 5, LET2-1, 6. LET2-2, 7. LET2-3, 8. CE1, 9. CET1-1, 10, CET1-2, 11. BE1, 12. BET. Lower panel: quantified the protein levels shown in upper panel using Odyssey infrared imaging system software version 3.0 from LI-COR Biosciences Company. (d) The levels of MYC or KLF4 were examined as the level of p53, and the levels of mir- 34b and c (with mature or pri-mir primers) as described in Figure 1. The value presented as mean + SD from three independent experiments. 1

2 Figure S2. OCT4 is a target of mir-34a-3p. (a) Left panel: the OCT4 levels measured from 293FT cells at different times after co-transfecting the plasmid (HA-OCT4-3 UTR or HA-OCT4 3 UTR) with or without a control vector. PCNA was used as an internal loading control. Right panel: quantified the protein levels shown in left panel using Odyssey infrared imaging system software version 3.0 from LI- COR Biosciences Company, CV: control vector. The 48 h points (in red frame) were chosen for the mir-34a vector transfection experiments. (b) The levels of mir-34a-3p measured using qrt-pcr from 293FT cells at different times after transfected with the plasmid encoding mir-34a or vector alone (control vector). The data are calibrated with internal control RNA, RNU48, and are the mean + SD from three independent experiments. (c) mir-34a targets OCT4 in human epithelial cells. LE1 or LET1-1 cells were transfected with either control RNAs or mir-34a mimics. Left panel: Western blot data: at 48 h after transfection, the cells were collected and whole cell lysates were prepared for a western blot assay. ctin was used as an internal loading control; Right panel: quantified the protein levels shown in left panel using Odyssey infrared imaging system software version 3.0 from LI-COR Biosciences Company. Similar results were obtained from two independent experiments. 2

3 Figure S3. mir-34a decreased the OCT4 level in mouse embryo stem cells. (a) The mir-34a-3p level in mouse embryo stem cells that were transfected with either control RNA or mir-34a mimic. The mir- 34a-3p levels were measured at 48 h after transfection using a real-time PCR approach in triple sets and the data were calibrated with internal control RNA, Sno202. (b) Immunoblots of whole cell lysates from mouse embryo cells measured at 24 h or 48 h after transfection with control RNA or the mir-34a mimics. ctin was used as an internal loading control. (c) Photomicrographs of mouse embryo stem cells at 48 h after transfection with either control RNA or mir-34a mimics. The cells were incubated with the primary antibody anti-ssea1 (StemCell Technologies Inc) and then with the FITC-conjugated second antibody (Millipore). 3

4 Figure S4. The relationship among OCT4, p53 and p63. (a) The effects of p53, p63 or OCT4 silencing on the expression of p53, p21, p63 or OCT4 in human epithelial cells. Upper panel, Western blot: human lung epithelial cells, LE1, and the transformed counterparts, LET1-1, were transfected with control RNA, OCT4 sirna, p53 sirna or p63 sirna. The cells were collected at 48 h after transfection and whole cell lysates were prepared for a western blot assay. ctin was used as an internal loading control. Bottom panel, the protein levels (p53, p21, p63 and OCT4) shown in the upper panel were quantified using Odyssey infrared imaging system software version 3.0 from LI-COR Biosciences Company. Similar results were obtained from two independent experiments. (b) 293FT cells were transfected with the plasmid with or without HA-OCT4. The levels of p53 or p63 in HA-OCT4 plasmid transfected cells were calculated as the ratio of that in empty vector transfected cells. Data shown are the mean + SD from three independent experiments. (c) 293FT cells were transfected with the plasmid with or without HA-OCT4. The levels of p63 with the TA-p63 TA-p63 or TA-p63 specific probes were examined using the real time PCR approach. 4

5 Figure S5. Outline of plasmid containing the promoter region of p53 or p63. (a) Outline of plasmid constructs containing partial of the promoter region of p63 for the luciferase assay. WT, containing the wild type of potential binding site of OCT4, mut 1-3: different mutation deleted the different potential binding site of OCT4. (b) The strategy for constructing OCT4-nonexpression plasmid. 293FT cells were transfected with OCT4 expression or nonexpression plasmid. The cells were collected at 48 h after transfection and whole cell lysates were prepared for the Western blot assay. ctin was used as an internal loading control. (c) Outline of plasmid constructs containing the promoter region of p53 for the luciferase assay. WT, containing the wild type of potential binding site of OCT4, mut 1-3: different mutation without the potential binding site of OCT4 either at N-terminal ( ) or at C-terminal ( ). 5

6 Figure S6. MiR-34a-3p targeting OCT4 affects cell functions. (a) The image of the GFP signals was detected from transformed human epithelial cells: LET1-1 or LET1-2 cells (Supplementary Table 1) that were stably expressed with GFP vector alone, the vector encoding GFP fused p53-shrna, p63-shrna or OCT4-shRNA. (b) The levels of mir-34a were measured by the qrt-pcr assay from LET1-1 or LET1-2 cells that were stably expressed with GFP vector alone, the vector encoding GFP-fused p53- shrna, p63-shrna or OCT4-shRNA. The error bars presented as mean + SD from three independent experiments. 6

7 Table S1. Description of human transformed cell lines used in this study Cell lines Description Abbreviation Source HBE3KT Immortalized human bronchial epithelial cells (HLEC) LE1 UT Southwestern University 3 HBE3KT-0.25F Fe transformed HLEC LET1-1 UT Southwestern University HBE3KT-0.5F Fe transformed HLEC LET1-2 Emory group generated transformed HBE3KT BEP2D R30TIL R30T3S R30T5S HCECICT Immortalized human bronchial epithelial cells -particle transformed BEP2D -particle transformed BEP2D -particle transformed BEP2D Immortalized human colon epithelial cells (HCEC) LE2 Columbia University 2 LET2-1 Columbia University 2 LET2-2 Columbia University 2 LET2-3 Columbia University 2 CE UT Southwestern University 4 HCECICTRPA Fe transformed HCEC CET1 UT Southwestern University 1 HCECICTRPA1309 Fe transformed HCEC CET2 UT Southwestern University 1 184B5 Immortalized human breast epithelial cells (HBEC) BE NASA Johnson Space Center 5 184B5F5/C3 Fe transformed 184B5 BET NASA Johnson Space Center 5 7

8 Table S2. Primers used in this study Primer Name Sequence 5'-3' Remark OCT4 GGTTGAGTAGTCCCTTCGC Expression form Forward (F) OCT4 Reverse (R) CACATCGGCCTGTGTATATC Expression form OCT4 (F) TTCAGTCAACATTTAATGATGCT pseudogene 1 OCT4 (R) GCCTGGTGAAATGAGCAATT pseudogene 1 OCT4 CGA CCA TCT GCC GCT TTG For all forms RT-PCR (F) OCT4 GCC GCA GCT TAC ACA TGT TCT For all forms RT-PCR (R) GC91- HA-OCT4-F CGCAGAATTCAAATGGCGGGACACCTGGCTTCGGATTTCGCC TTCTCG GC87-R TACCGCGGCCGCTCAGTTTGAATGCATGGGAGAG Without 3 UTR GC92-R TACCGCGGCCGCTAAGTGTGTCTATCTACTGTGTCCCAGGC With 3 UTR GC93-F GAAGGGCAAGCGATCAAGCAGCGACTATGCACAACGAGAGG With 3 UTR GC94-R CCTCTCGTTGTGCATAGTCGCTGCTTGATCGCTTGCCCTTC With 3 UTR GC153-HA-OCT4- AACACAAAGGGTGGGGGCAGGAGGGAAGGTGAAGTTCAATG Mutant 3 UTR Mu-F GC154-HA-OCT4- CATTGAACTTCACCTTCCCTCCTGCCCCCACCCTTTGTGTT Mutant 3 UTRMu-R GC147-OCT4- ACCGCTCGAGTGAGGTGCCTGCCCTTCTAGGAATG Wild type 3 UTR-F GC92-OCT4- TACCGCGGCCGCTAAGTGTGTCTATCTACTGTGTCCCAGGC Wild type 3 UTR-R GC148-OCT4- GGCAGGGGAGTTTGGGGCAAGAGGGAAGGTGAAGTTCAATG Mutant 3 UTRMu-F GC149-OCT4- CATTGAACTTCACCTTCCCTCTTGCCCCAAACTCCCCTGCC Mutant 3 UTRMu-R GC156-p53F1 AAACGGTACCGTCCGTCCACCACTTTATCCCAGCAC GC158-p53R1 AAGGAAGATCTGGACCCAGGTTTATTGTCCCCC GC159-p53F2 AAACGGTACCGAAGTTCTCAGGTTGGGTGCTG GC161-p53R2 AAGGAAGATCTCATGGAAACGTAAGCCTTAATGAGGATATAG GC160-p53F3 AAACGGTACCCAAGAAGTTCTCAGGTTGGGTGC GC162-p53R3 AAGGAAGATCTGTACATGGAAACGTAAGCCTTATGAGGATA GC163-p53MUT1 AAACGGTACCAGGCAGCTGGGAGAAGCTGTAG GC164-p53MUT2 AAACGGTACCCAGCTGGGAGAAGCTGTAGTTCC GC165-p53MUT3 AAGGAAGATCTATTCCTGAGTGCCTATATCAGTGCTGG GC166-p53MUT4 AAGGAAGATCTCCTGAGTGCCTATATCAGTGCTGG GC194-p53(7)F AGTTCTCAG GTTGGGTGCTG For ChIP GC195-p53(7)R ATTGTC CCC CATCTTCCTTT For ChIP GC118-p53-F GTTCCGAGAGCTGAATGAGG For real-time PCR GC119-p53-R TCTGAGTCAGGCCCTTCTGT For real-time PCR 8

9 Primer Name Sequence 5'-3' Remark GC116-TAp63-F TGTATCCGCATGCAGGACT For TAp63 GC117-TAp63-R CTGTGTTATAGGGACTGGTGGAC For TAp63 GC114-DNp63-F GAAAACAATGCCCAGACTCAA For ΔNp63 GC115-DNp63-R TGCGCGTGGTCTGTGTTA For ΔNp63 GC237-TAp63αF CTTAGCGAGGTTGGGCTGTTCATCATG For TAp63α GC239-TAp63αβR GATGAGAAGGGGAGGAGAATTCGTGGA For TAp63α GC238TAp63βF CAGCATTGTCAGGATCTGGCAAGTCT For TAp63β GC239TAp63αβR GATGAGAAGGGGAGGAGAATTCGTGGA For TAp63β GC240TAP63γF CAAACCGATCAGTGTACCCATAGAGC For TAp63γ GC241Tap63γR CCTTTGAGCAGTTGGGTCTCTGAG For TAp63γ GC131-p63F2 GC132-p63R2 GC139-p63Mu1F GC140-p63Mu1R GC141-p63Mu2F GC142-p63Mu2R GC143-p63Mu3F CGGGGTACCAAAGACCTCAGGAGGACCAAGAACAAG TTCT ATTAAGATCTCCTAAAGTTACTAAGCTAAGAGGAACA GAAATTAAATGGC GCAGCTATAATAAGAGTGAATTACTGATACAGTTGAC ACTCAGAAACCATGGCATCAATGAGCAAAAAAAATC GATTTTTTTTGCTCATTGATGCCATGGTTTCTGAGTGT CAACTGTATCAGTAATTCACTCTTATTATAGCTGC GAAAGTGCATGAAGGGGAGCAGTTGTAAAATAATTT GTGAGAAGAATTTGTATCATAGACACCTGAGTTTG CAAACTCAGGTGTCTATGATACAAATTCTTCTCACAA ATTATTTTACAACTGCTCCCCTTCATGCACTTTC CCTAAGAGAATAAGGATGAACTATAAATATAAGAAG CAATTATGGTAATTATACTTATTTTTCACTTGATCGTG TATGGTTGCATGC GC144-p63Mu3R GCATGCAACCATACACGATCAAGTGAAAAATAAGTA TAATTACCATAATTGCTTCTTATATTTATAGTTCATCC TTATTCTCTTAGG GC188-p63 CAT GAA GGG GAG CAG TTG TAA For ChIP (5)F GC189-p63 CCT AGG AGC CAA GAA CAT GC For ChIP (5)R GAPDH-RT-F ATG GAA ATC CCA TCA CCA TCT T Intern control for real time PCR GAPDH-RT-R CGG CCC ACT TGA TTT TGG Intern control for real time PCR 9

10 Table S3. EMSA oligos Oligo Name Sequence 5'-3' Remark GNEMSAp63-4 CAACATGAATATATGAATATATCT p63 promoter OCT4 binding site 1 GNEMSAp63-5 TTTCATGAATGTATGAATGTAAGT p63 promoter OCT4 binding site 2 GNEMSAp63-6 TAATATGATTGCATGATTGCCACT p63 promoter OCT4 binding site 3 GNEMSAp53-7 ACTGAGAAATCGTGAGAAATCGAG p53 promoter OCT4 binding site 1 GNEMSAp53-8 ATACAACAATGAACAACAATGAAT p53 promoter OCT4 binding site 2 Supplementary References 1 Eskiocak U, Kim SB, Roig AI, Kitten E, Batten K, Cornelius C et al. CDDO-Me protects against space radiation-induced transformation of human colon epithelial cells. Radiat Res : Hei TK, Piao CQ, Willey JC, Thomas S, Hall EJ. Malignant transformation of human bronchial epithelial cells by radon-simulated alpha-particles. Carcinogenesis : Ramirez RD, Sheridan S, Girard L, Sato M, Kim Y, Pollack J et al. Immortalization of Human Bronchial Epithelial Cells in the Absence of Viral Oncoproteins. Cancer Res : Roig AI, Eskiocak U, Hight SK, Kim SB, Delgado O, Souza RF et al. Immortalized Epithelial Cells Derived From Human Colon Biopsies Express Stem Cell Markers and Differentiate In Vitro. Gastroenterology : e Yang T, Georgy K, Tavakoli A, Craise L, Durante M. Radiogenic Transformation of Human Mammary Epithelial Cells In Vitro. Radiat Onc Invest :