a

b

cR

ela

tive

mR

NA

exp

ress

ion

WA

01

WA

07

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CR

L-2

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7

i14

i15

i16

i17

i18

i19

i20

i21

i22

i23

i24

i25

Nanog

133p53

p62/SQSTM1

LC3-I

β-actin

(Baf A1)

i20

(iPSC)

LC3-II

1 1.3

1 4.8

37 kDa

50 kDa

15 kDa

37 kDa

WA

01

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CR

L-2

097

i14

i15

i16

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i25

133p53

GAPDH

3.3 2.0 1.9 1 2.3 2.8 2.3 3.4 3.6 3.4 3.4 3.0 3.1 2.5 3.4 3.2

Supplementary Figure S1 ∆133p53 expression in human pluripotent stem cells. (a) qRT-PCR analysis of Nanog mRNA

expression. The expression values in iPSC and ESC lines are shown in relative to CRL-2097 (defined as 1.0). (b) RT-PCR

analysis of ∆133p53 mRNA expression. GAPDH was an internal control to normalize the mRNA expression levels of

∆133p53 in the quantitative analysis. The expression values in iPSC and ESC lines are shown in relative to CRL-2097

(defined as 1.0). (c) Inhibition of autophagy in iPSC. iPSC (i20) were untreated (-) or treated with bafilomycin A1 (+, 100 nM

for 4 h) and examined in western blot for indicated proteins. The inhibition of autophagy was confirmed by increased LC3-II.

-actin was a loading control for normalization. The relative expression levels of 133p53 and p62/SQSTM1 are shown

below the images. (d) Yamanaka factors (YF) by themselves have a minimal effect on ∆133p53 expression. CRL-2097 was

transduced with the retroviral pMXs construct for expression of Oct-4, Sox-2, Klf-4 and c-Myc (+) or control vector (-),

followed by western blot before used in the iPSC reprogramming protocol. An iPSC line (i20) was examined in parallel.

d

a b

Rela

tive m

RN

Aexp

ressio

n

p21WAF11.4

1.2

1

0.8

0.6

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Aexp

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n

PAI-11.4

1.2

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0

BAX1.2

1

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0.6

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PUMA1.2

1

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p53R21.2

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tive m

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IGFBP71.2

1

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0

Vec ∆133

Rela

tive m

RN

Aexp

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n

Vec ∆133

miR-34a1.2

1

0.8

0.6

0.4

0.2

0

CRL-2097

human

fibroblasts

Vec ∆133

∆133p53

-actin

37 kDa

37 kDa

Supplementary Figure S2 Overexpression of ∆133p53 in human fibroblasts represses a subset of p53-inducible

genes. Normal human fibroblasts (CRL-2097 strain) were retrovirally transduced with an overexpression vector of

∆133p53 and its empty vector control (Vec). (a) Western blot analysis confirming ∆133p53 overexpression. (b)

mRNA expression of p53-inducible genes. The qRT-PCR analyses were performed as in Figures 2 and 3b. The

expression levels in ∆133p53-overexpressing cells are shown in relative to those in Vec control (defined as 1.0).

Means ± S.D. are shown, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired two-tailed

Student’s t test.

a b

c

d

Supplementary Figure S3 Full-length p53 (FL-p53) contributes to the expression of BAX, PUMA and p53R2 in human

fibroblasts with overexpressed ∆133p53 and in iPSC with abundant expression of endogenous ∆133p53. BJ with

overexpressed ∆133p53, CRL-2097 with overexpressed ∆133p53 and an iPSC line (i20) were transfected with siRNA

targeting the N-terminal region of p53 (amino acids 49-54, which are not present in ∆133p53) and control siRNA (C),

followed by isolation of protein and RNA samples after 3 days of culture. (a) Western blot analysis confirming the

knockdown of FL-p53. (b-d) qRT-PCR analyses of mRNA expression of BAX (b), PUMA (c) and p53R2 (d). Expression

data are means ± S.D. (n = 3) and shown in relative to control-transfected cells (defined as 1.0) in each cell line. ***P <

0.001, ****P < 0.0001, as determined by unpaired two-tailed Student’s t test.

a

b

c

Vec ∆133

CRL-2097 fibroblasts

(5 104 cells per well)

46,XY (normal)

1 2 3 4 5

6 7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 X Y

∆133p53

-actin

Doxy (-) (+)

BJ fibroblasts

+ inducible ∆133

37 kDa

37 kDa

Supplementary Figure S4 Overexpression of ∆133p53 enhances iPSC reprogramming. (a) Representative result

of iPSC generation. The iPSC colonies generated from CRL-2097 fibroblasts with control vector (Vec) or

overexpressed ∆133p53 (5 104 cells per well in an experiment shown in Figure 4c) were stained with alkaline

phosphatase. (b) Western blot analysis demonstrating inducible expression of ∆133p53 by addition of doxycycline

(Doxy) in BJ fibroblasts. The cells were used in Figure 4d. (C) G-banding karyotype analysis of iPSC generated

from ∆133p53-overexpressing fibroblasts. Six iPSC lines from CRL-2097 with overexpressed ∆133p53 (Figure 4c)

were examined and all of them were found to have normal chromosomes. A representative normal karyotype (46,

XY) is shown.

Supplementary Figure S5 Examples of microsatellite analysis in the iPSC lines (Bi133-1 and Bi53KD are shown)

and the original fibroblasts (BJ is shown). Five different microsatellite loci were simultaneously PCR-amplified and

analyzed by a fluorescence-based capillary electrophoresis. Size standards (ROX-labeled, shown as blue peaks) and

microsatellite peaks (FAM-labeled, shown as black peaks) are indicated with their sizes (bp). Bi133-1 and Bi53KD

show the identical profiles to BJ.

Supplementary Figure S6 Mitochondrial DNA (mtDNA) copy numbers in the iPSC lines and the

original fibroblasts. The qRT-PCR-based determination of mtDNA copy numbers, normalized with

nuclear DNA amounts, was performed using Human Mitochondrial DNA Copy Number Assay kit

(Detroit R&D, Detroit, MI) following the supplier’s protocol. The mtDNA copy numbers in the

iPSC lines are shown as relative values to those in their original fibroblasts (BJ, defined as 1.0, and

its derived iPSC lines on the left; and CRL-2097, defined as 1.0, and its derived iPSC lines on the

right). Data are means ± S.D. (n = 3). *P < 0.05, **P < 0.01, as determined by unpaired two-tailed

Student’s t test.

a

b

c

d

e

f

g

h

Supplementary Figure S7 ∆133p53 protein

expression and p53 target gene expression in

iPSC lines generated from ∆133p53-

overexpressing CRL-2097 (Ci133-1, Ci133-2,

Ci133-3 and Ci133-4) and iPSC lines from

vector-transduced control cells (CiV1, CiV2

and CiV4). (a) Western blot analysis of ∆133p53 protein expression. The expression levels in iPSC lines are shown in relative to CRL-2097. (b-h) qRT-PCR analyses of mRNA expression of p21WAF1 (b), PAI-1 (c), IGFBP7 (d), miR-34a (e), BAX (f), PUMA (g) and p53R2 (h). Expression data are means ± S.D. (n = 3) and shown in relative to CRL-2097 (defined as 1.0). **P < 0.01, ***P < 0.001, ****P < 0.0001, as determined by unpaired two-tailed Student’s t test.

a b

∆133p53

-actin

ATG5-ATG12

ATG7

Beclin-1

MDAH041-/-

(p53-null)

1 1.9

1 1.8

1 1.5

Vec ∆133

50 kDa

75 kDa

50 kDa

37 kDa

37 kDa∆133p53

Vec ∆133

-actin

ATG5-ATG12

ATG7

Beclin-1

MRC-5 human

fibroblasts

1 1.7

1 1.3

1 2.8

50 kDa

75 kDa

50 kDa

37 kDa

37 kDa

Supplementary Figure S8 Overexpression of ∆133p53 upregulates autophagy-mediating proteins. Normal human

fibroblasts MRC-5 (a) and p53-null immortalized human fibroblasts MDAH041-/- (b) were retrovirally transduced

with an overexpression vector of ∆133p53 and its empty vector control (Vec). Western blot analysis was performed

to examine the expression of Beclin-1, ATG7 and ATG5-ATG12 conjugate (detected by anti-ATG5 antibody). The

relative expression levels of these autophagy-mediating proteins (normalized with β-actin) are shown below the

images.

Supplementary Table S1. iPSC lines established in this study. iPSC lines Fibroblasts Exogenous133p53 Method Experiment Karyotype Teratoma

BiC1 BJ Untransduced mRNA n.d. n.d.Bi133(-) BJ Not induced mRNA Fig.4d, Doxy(-) n.d. n.d.Bi133-1 BJ (+) mRNA Fig.4d, Doxy(+) 46,XY n.d.Bi133-2 BJ (+) mRNA Fig.4d, Doxy(+) n.d. n.d.Bi133-3 BJ (+) mRNA Fig.4d, Doxy(+) 46,XY n.d.Bi53KD BJ (-), p53 KD* mRNA 46,XY n.d.

CiC1 CRL-2097 Untransduced mRNA 46,XY BenignCiC2 CRL-2097 Untransduced mRNA n.d. n.d.CiV1 CRL-2097 Vector control mRNA Fig.4c, Vec n.d. BenignCiV2 CRL-2097 Vector control mRNA Fig.4c, Vec 46,XY n.d.CiV3 CRL-2097 Vector control mRNA Fig.4c, Vec n.d. n.d.CiV4 CRL-2097 Vector control mRNA Fig.4c, Vec n.d. Benign

Ci133-1 CRL-2097 (+) mRNA Fig.4c, 133 n.d. n.d.Ci133-2 CRL-2097 (+) mRNA Fig.4c, 133 46,XY*** n.d.Ci133-3 CRL-2097 (+) mRNA Fig.4c, 133 46,XY Benign****Ci133-4 CRL-2097 (+) mRNA Fig.4c, 133 46,XY n.d.Ci133-5 CRL-2097 (+) mRNA Fig.4c, 133 46,XY Benign****

* Retroviral knockdown of p53.** From untransduced or p53-knocked-down fibroblasts under conditions identical to Figure 4c and 4d.*** Shown as an example in Supplementary Figure S4c.**** Shown in Figure 4e.

Supplementary Table S2. Microsatellites and mitochondrial DNA (mtDNA) sequences in fibroblasts and iPSC lines.

Microsatellite loci, allele sizes (bp)* mtDNA D-loop**Cells ACTC BAT26 D5S107 D5S406 D13S153 nt.152 T/C*** nt.494 (C)n****

BJ 70, 88 119 127, 143 167, 179 210, 212 C (C)4BiC1 70, 88 119 127, 143 167, 179 210, 212 C (C)4

Bi133(-) 70, 88 119 127, 143 167, 179 210, 212 C (C)4Bi133-1 70, 88 119 127, 143 167, 179 210, 212 C (C)4Bi133-2 70, 88 119 127, 143 167, 179 210, 212 C (C)4Bi133-3 70, 88 119 127, 143 167, 179 210, 212 C (C)4Bi53KD 70, 88 119 127, 143 167, 179 210, 212 C (C)4

CRL-2097 72, 86 118 167, 175 216, 220 T (C)5CiC1 72, 86 118 167, 175 216, 220 T (C)5CiC2 72, 86 118 167, 175 216, 220 T (C)5CiV1 72, 86 118 167, 175 216, 220 T (C)5CiV2 72, 86 118 167, 175 216, 220 T (C)5CiV3 72, 86 118 167, 175 216, 220 T (C)5CiV4 72, 86 118 167, 175 216, 220 T (C)5

Ci133-1 72, 86 118 167, 175 216, 220 T (C)5Ci133-2 72, 86 118 167, 175 216, 220 T (C)5Ci133-3 72, 86 118 167, 175 216, 220 T (C)5Ci133-4 72, 86 118 167, 175 216, 220 T (C)5Ci133-5 72, 86 118 167, 175 216, 220 T (C)5

* All five microsatellite loci in all iPSC lines were identical to those in their original fibroblasts.** mtDNA D-loop nucleotides 128-599 were sequenced. No homoplasmic or heteroplasmic mutation was found in any iPSC lines.*** A single nucleotide polymorphism at nucleotide position 152 (T or C).**** Different numbers (n) of C-stretch starting at nucleotide position 494.

Supplementary Table S3. Statistics of mapping, coverage and depth in whole exome sequencing.Cells BJ BiC1 Bi133(‐) Bi133‐1 Bi133‐3 Bi53KD CRL‐2097 CiV2 CiV4 Ci133‐2 Ci133‐3

Total number of clean reads 98,691,294 117,987,342 92,607,280 92,357,458 95,390,422 87,820,306 97,762,896 100,034,776 97,192,998 90,781,100 81,314,668Mapped to the reference genome hg19 99.94% 99.70% 99.41% 99.63% 99.73% 98.98% 99.95% 99.82% 99.51% 99.62% 99.78%

Total effective yield (Mb) 12911.21 14941.45 12073.94 12049.06 12378.42 11009.64 12815.1 12940.45 12622.36 11810.2 10696.81Average sequencing depth on target 134.39 156.91 128.32 130.91 133.43 115.48 135.61 137.8 132.56 126.54 114.75

Coverage of target region 99.9% 99.9% 99.9% 99.9% 99.9% 99.9% 99.9% 99.9% 99.9% 99.9% 99.9%Target covered with at least 10x 99.7% 99.7% 99.6% 99.6% 99.6% 99.6% 99.6% 99.7% 99.6% 99.6% 99.5%

Supplementary Table S4. Missense, premature stop and frameshift mutations in iPSC lines§ 

BiC1 PPP1R37 CCDC178* ARHGAP30 DHX33* PIK3C2A TTN† EIF4A2 CDH10 IGFN1 CCDC108ASIC3* GGT1 ZNF200 CSMD3 MUC16 DPP3 PARP2* RPGRIP1L PCDHA8 SBF2

Bi133(‐) KIF21B DENND1C* MGA OR56A1Bi133‐1 DNAH7 LRP12 KMT2B* SFMBT2 ZNF598 CMKLR1 LETM2 GPR179* RP1L1 ZNF483

APOL6 CPEB2 OTOG**†Bi133‐3 APOB* LCP1 KNG1 SCLT1 IRF4 TRH SCN5A SPDYE5 ZNF211** LCA5L

SEH1L RIMKLB SMURF2 EXOC1 SLC7A13Bi53KD CPSF2 PTPRO* CPNE8 E4F1 NAALADL2 DHX29 DMTF1 MATK GSAP ATP2A3

GSDMA ARHGAP32 SPTBN5* CD1B ASTN2 GABRA5 CEACAM1 REST ZNF148 PRAMEF1TTLL11 LILRB2 CPAMD8 ABL2 REXO1** ANAPC5**

CiV2 MYO5A* VWA3B SLC5A1 TRAF5 KRT76 TTN† PPP1R12C** TOX3* SGCECiV4 CXorf22 SP140L CCDC176 UBA7 DNAJB8* ZNF528 DSP MYO15A KIAA1324L

Ci133‐2 INS GPR180 SLC27A6 ERN1 LGR5 DNAH8 TET1 ANLN HEG1 RYR2ZNF407 ZCCHC3 OTOG† PACS2

Ci133‐3 OTUD7A CSF2RB TSHZ1 ADGB** GALNT16 ADK§ Listed are genes with mutant allele frequency of 0.30 or greater.   Missense (unmarked), premature stop (*) and frameshift (**) mutations are shown.† TTN and OTOG are shared by two iPSC lines, which were derived from different fibroblasts. All others are unique to a single iPSC line.