The p53-puma axis suppresses ipsc generation

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1 The p53-puma axis suppresses ipsc generation Yanxin Li, Haizhong Feng, Haihui Gu, Dale W. Lewis, Youzhong Yuan, Lei Zhang, Hui Yu, Peng Zhang, Haizi Cheng, Weimin Miao, Weiping Yuan, Shi-Yuan Cheng, Susanne M. Gollin, and Tao Cheng 1

2 Supplementary Figure S1. Morphology of induced cell colonies during reprogramming. Representative images of colonies at reprogramming day 5, day 14 compared to the images of newly formed third passage ipscs of each genotype. Bars, 50 μm. 2

3 Supplementary Figure S2. Expression of SSEA1 and Nanog in various genotype ipscs. Representative images of SSEA1, Nanog expression and DAPI staining in ipscs at day 21 after retroviral transduction. Bars, 50 μm. Results represent three independent experiments with similar results. 3

4 Supplementary Figure S3. Effect of introduction of PUMA, p21, p53 genes back into WT and the corresponding knockout MEFs. Effect of ectopic expression of PUMA, p21, p53 WT, or p53 S58A mutant in WT (a) or different knockout MEFs on ipsc generation (c to e). Various MEFs were transduced with retroviruses encoding PUMA, p21, p53 WT, mutant p53 S58A cdna or an empty vector together with 4F. On day 21 after transduction, the numbers of SSEA1 + and Nanog + colonies were counted and normalized to that of the vector control. The Western blots in b demonstrate the expression of p53, PUMA and p21 in WT MEFs with ectopic expression of PUMA, p21, p53 WT, p53 S58A or an empty vector. Error bars in a, c to e, s.e.m.. Statistical significance was tested using the Oneway ANOVA with Newman-Keuls post-test (a and c). Statistical significance was tested using a 4

5 paired two-way Student s t test (d and e). *, P < 0.05; **, P < 0.01; ***, P < Results in a, and c to e represent three independent experiments with similar results. Supplementary Figure S4. Characterization of the cell lines differed in morphology. (a). The morphorlogy of induced cell colonies at Grade I, II and III observed by phase contrast and expression of Oct4, Nanog, Sox2 and SSEA1 in the representative colonies from each Grade. Bars, 50 μm. Inserts, using an anti-igg antibody. (b). Percentage of Grade I induced cell lines with various genotypes. Error bars, s.e.m.. Statistical significance was tested using the One-way ANOVA with Newman-Keuls post-test (b). ** P <0.01. Results in b represent three independent experiments with similar results. 5

6 Supplementary Figure S5. Effect of heterozygous p53, p21 and PUMA on ipsc generation. AP-stained positive colonies derived from PUMA -/-, p21 -/-, p53 -/-, PUMA +/-, p21 +/- and p53 +/- MEFs transducted with 4F (a) with 3F in the absence of Myc (b) relative to that from WT MEFs at reprogramming day 14. Error bars in a and b, s.e.m.. Statistical significance was tested using the One-way ANOVA with Newman-Keuls post-test (a and b). **, P <0.01. Results in a and b represent three independent experiments with similar results. 6

7 Supplementary Figure S6. Effect of hpuma on human ipsc generation. a, Western blotting analysis of PUMA protein expressions, β-actin used as a control. b, Effect of the relative reprogramming efficiencies of hpuma on human ipsc generation. Human fibroblasts were firstly infected with retroviruses encoding hoct4, hsox2, hklf4, and hc-myc (four factors, 4F) and then infected with hpuma shrna or GFP shrna. At day 30, cells were stained to detect alkaline phosphatase (AP) activity. Data were normalized to the numbers of AP + colonies in vector control. Error bars, s.e.m.. Statistical significance was tested using the One-way ANOVA with Newman-Keuls post-test (b). * P <

8 Supplementary Figure S7. Interphase and anaphase brigde analysis in MEF and reprogrammed cells. a. Representative images of anaphase and interphase bridges of WT ipscs and WT MEFs, respectively. Bars, 10 μm. b and c, Percentage of interphase and anaphase bridges of various genotype ipscs (b) and MEFs (c). The reprogrammed cells at day 12 and MEFs at passage 3 were harvested. Approximately 50,000 cells per sample were reseeded on a 22 x 22 mm glass coverslip. Cells were allowed to adhere and proliferate for 16 h prior to fixation and staining with DAPI. Anaphase and interphase bridges events were scored by studying approximately 1,000 cells from each of the five genotypes of ipsc and MEF cultures. The images shown were taken with 100 x oil objective. 8

9 Supplementary Figure S8: Full scans of western data. Rectangles delimit cropped areas used in the indicated figures. 9

10 Supplementary Table S1. Generation of chimeras from ipscs. Microinjection in ICR blastocysts ID # of ipsc clones Genotype Blastocysts injected Cells injected Pups born Chimeras, Sex (% Pigmentation) ES PUMA -/- 40 7~11 3 1M (10%), 1M (1%) N41(c-Kit + ) PUMA -/ ~ M (50%), 1M (70%), N44 PUMA -/- 98 7~ M (80%), 1M (40%), 1F (20%) N34 PUMA -/- 64 7~11 9 1F (20%) N18 PUMA -/ ~ , 2 death N36 PUMA -/- 70 7~ N1 p53 -/- 73 7~ N4 p53 -/- 96 7~ N3 p53 -/- 96 7~ N2 p53 -/- 66 7~ N21 p21 -/ ~11 10 All death N4 p21 -/- 97 7~11 8 0, 4 death, N11 p21 -/ ~

11 Supplementary Table S2. Chromosome instability in the four ipsc genotypes (Set #1). ipsc genotype ips cell line Ploidy level Chromosome # range Chromosome Gain a Chromosome loss Structural Alterations Clonal Differences from WT WT N404 Diploid (2n) mar 6 WT N2GFP Near-tetraploid (4n) mar 5 WT N413 Diploid (2n) mar 7 p53 -/- N13 Near-tetraploid (4n) Y, del(2) del(15) 23 p53 -/- N4 Near-tetraploid (4n) Y, del(2) 20 p53 -/- N14 Near-tetraploid (4n) del(2) 28 p21 -/- N2 Diploid (2n) , del(15) 3 p21 -/- N21 Diploid (2n) del(15) 9 p21 -/- N16 Diploid (2n) , +12, del(15) 19 PUMA -/- N34 Diploid (2n) PUMA -/- N32 Diploid (2n) add(14) 6 PUMA -/- N19 Diploid (2n) Instability Events b a, The number of chromosomal gains in the 20 cells analyzed. Likewise, loss and structural alterations are represented in the same manner. Figure 4d and associated statistical analyses of chromosomal instability are based on the sum of chromosome gain, loss and structural alterations in each cell lines. b, Instability events are defined as the number of different cellular events necessary to induce the numerical and structural aberrations observed (instability is represented by the first occurrence of a particular chromosomal alteration, assuming that the additional occurrences of the same alteration are in daughter cells. 11

12 Supplementary Table S3. Chromosome instability in the four MEF genotypes (Set #1). MEFs Ploidy level Chromosome # range Chromosome Gain a Chromosome loss Structural Alterations Clonal differences from WT Instability Events b WT Diploid-2n(15) Tetraploid-4n (5) p53 -/- Near-Tetraploid-4n(19) Near-Hexaploid-6n (1) del(2) 7 p21 PUMA -/- -/- Diploid-2n (7) Near-Tetraploid-4n (13) Diploid-2n(16) Near-Tetraploid-4n (3) Near-Hexaploid-6n (1) a, The number of chromosomal gains in the 20 cells analyzed. Likewise, loss and structural alterations are represented in the same manner. Figure 4 and associated statistical analyses of chromosomal instability are based on the sum of chromosome gain, loss and structural alterations in each cell lines. b, Instability events are defined as the number of different cellular events necessary to induce the numerical and structural aberrations observed (instability is represented by the first occurrence of a particular chromosomal alteration, assuming that the additional occurrences of the same alteration are in daughter cells. 12

13 Supplementary Table S4. Chromosome instability in the four ipsc genotypes (Set #2). ips genotype WT p53 -/- p21 -/- ips cell line Ploidy level Chromosome # range Chromosome Gain a Chromosome loss Structural Alterations Clonal Differences from WT M3 Near-Diploid (2n) M4 Near-Diploid (2n) mar 4 M5 Near-Diploid (2n) M5 M6 M7 Near-Diploid (2n) (10) Near-tetraploid (4n)(10) Near-Diploid (2n) (11) Near-tetraploid (4n)(9) Near-Diploid (2n) (15) Near-tetraploid (4n)(5) Instability Events b del(2) del(2) 17 M3 Near-Diploid (2n) M4 Near-Diploid (2n) M6 Diploid (2n) PUMA -/- M4 Diploid (2n) M3 Diploid (2n) M5 Near-Diploid (2n) a, The number of chromosomal gains in the 20 cells analyzed. Likewise, loss and structural alterations are represented in the same manner. Figure 4d and associated statistical analyses of chromosomal instability are based on the sum of chromosome gain, loss and structural alterations in each cell lines. b, Instability events are defined as the number of different cellular events necessary to induce the numerical and structural aberrations observed (instability is represented by the first occurrence of a particular chromosomal alteration, assuming that the additional occurrences of the same alteration are in daughter cells. 13

14 Supplementary Table S5. Chromosome instability in the four MEF genotypes (Set #2). MEFs Ploidy level Chromosome # range Chromosome Gain a Chromosome loss Structural Alterations Clonal differences from WT Instability Events b WT Diploid-2n(14) Near-Tetraploid-4n (6) p53 -/- Diploid-2n(10) Near-Tetraploid-4n (10) p21 -/- Diploid-2n (11) Near-Tetraploid-4n (9) PUMA -/- Diploid-2n(18) Near-Tetraploid-4n (2) a, The number of chromosomal gains in the 20 cells analyzed. Likewise, loss and structural alterations are represented in the same manner. Figure 4 and associated statistical analyses of chromosomal instability are based on the sum of chromosome gain, loss and structural alterations in each cell lines. b, Instability events are defined as the number of different cellular events necessary to induce the numerical and structural aberrations observed (instability is represented by the first occurrence of a particular chromosomal alteration, assuming that the additional occurrences of the same alteration are in daughter cells. 14

15 Supplementary Table S6. Chromosome instability in the four ipsc genotypes (Set #3). ipsc genotype WT p53 -/- p21 -/- ips cell line Ploidy level Chromosome # range Chromosome Gain a Chromosome loss Structural Alterations Clonal Differences from WT P7 Near-Diploid (2n) P8 Near-Diploid (2n) P9 Near-Diploid (2n) P8 Near-Diploid (2n)(10) Near-tetraploid (4n)(10) del(3) 11 P9 Near-Diploid (2n)(11) Near-tetraploid (4n)(9) del(4) 15 P10 Near-Diploid (2n)(15) Near-tetraploid (4n)(10) P5 Diploid (2n) P7 Diploid (2n) del(3) 9 P8 Diploid (2n) del(2) 7 Instability Events b PUMA -/- P9 Diploid (2n) add(3) 6 P8 Diploid (2n) P10 Diploid (2n) a, The number of chromosomal gains in the 20 cells analyzed. Likewise, loss and structural alterations are represented in the same manner. Figure 4d and associated statistical analyses of chromosomal instability are based on the sum of chromosome gain, loss and structural alterations in each cell lines. b, Instability events are defined as the number of different cellular events necessary to induce the numerical and structural aberrations observed (instability is represented by the first occurrence of a particular chromosomal alteration, assuming that the additional occurrences of the same alteration are in daughter cells. 15

16 Supplementary Table S7. Chromosome instability in the four MEF genotypes (Set #3). MEFs Ploidy level Chromosome # range Chromosome Gain a Chromosome loss Structural Alterations Clonal differences from WT Instability Events b WT Diploid-2n(16) Tetraploid-4n (4) p53 -/- Diploid-2n(13) Tetraploid-4n (7) p21 -/- Diploid-2n (15) Near-Tetraploid-4n (5) PUMA -/- Diploid-2n(16) Near-Tetraploid-4n (4) a, The number of chromosomal gains in the 20 cells analyzed. Likewise, loss and structural alterations are represented in the same manner. Figure 4 and associated statistical analyses of chromosomal instability are based on the sum of chromosome gain, loss and structural alterations in each cell lines. b, Instability events are defined as the number of different cellular events necessary to induce the numerical and structural aberrations observed (instability is represented by the first occurrence of a particular chromosomal alteration, assuming that the additional occurrences of the same alteration are in daughter cells. 16

17 Supplementary Table S8. Bridges as a measure of chromosome instability in the four each genotype of ipscs and MEFs. Cell type Total cell numbers Anaphase bridges Interphase bridges WT ipscs WT MEFs p53 -/- ipscs p53 -/- MEFs p21 -/- ipscs p21 -/- MEFs PUMA -/- ipscs PUMA -/- MEFs PUMA -/- / p21 -/- ipscs PUMA -/- / p21 -/- MEFs