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(A) the Level of PCBP4 Protein Was Measured in WT and P53-/- Mefs

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(A) the Level of PCBP4 Protein Was Measured in WT and P53-/- Mefs Figure S1 A MEFs B R T L SA βgeo pA R T L Targeting vector +/+ -/- p53 p53 E1 E2 Wild-type PCBP4 allele PCBP4 GM103F GM104F SA β pA Actin E1 geo E2 Targeted PCBP4 allele GM103R C Mice D MEFs E MEFs +/+ +/- -/- PCBP4 +/+ +/- -/- PCBP4 +/+ +/- -/- PCBP4 GM103F-GM103R GM103F-GM103R PCBP4 GM104F-GM103R GM104F-GM103R Actin β-hemoglobin β-hemoglobin F Male, 15 days Female, 15 days G 35 ) s 30 - / m - a 4 r 25 P * g ( B t C 20 h P + g / i + e 4 15 P w F, PCBP4+/+ (n=3) B y C 10 +/- d + F, PCBP4 (n=3) - / P / o - + -/- 4 4 B 5 F, PCBP4 (n=3) P P B B C C 0 P P 4 6 8 10 12 14 16 18 20 Age (weeks) H 50 I 50 ) ) s 40 s 40 * m m a a r r g g ( * ( 30 t 30 t h h g g i i e e w w 20 20 M, PCBP4+/+ (n=13) y y +/+ F, PCBP4 (n=10) d d M, PCBP4+/- (n=12) o o F, PCBP4+/- (n=8) B B 10 10 M, PCBP4-/- (n=3) F, PCBP4-/- (n=9) 0 0 20 23 26 29 32 35 38 41 44 47 20 23 26 29 32 35 38 41 44 47 Age (weeks) Age (weeks) Figure S1. Generation of PCBP4 knockout mice and MEFs. (A) The level of PCBP4 protein was measured in WT and p53-/- MEFs. (B) PCBP4 knockout strategy and primers used for genotyping. The PCBP4 gene was disrupted by insertion of a gene trap (rFRosaβgeo+1s) into the first intron. Primers GM103F and GM103R were used for genotyping wild-type PCBP4 allele; primers GM104F and GM103R were used for genotyping the null allele. (C) Genotyping of WT, PCBP4+/-, and PCBP4-/- mice. Top and middle panels represent wild-type and knockout alleles of PCBP4, respectively. Bottom panel: β-hemoglobin was measured as an internal control. (D) Genotyping of PCBP4 knockout MEFs. (E) The level of PCBP4 protein was measured in WT, PCBP4+/-, and PCBP4-/- MEFs. Actin was measured as an internal control. (F) Representative images of 15-day-old WT and PCBP4-/- male and female littermates. (G) The body weight of WT, PCBP4+/-, and PCBP4-/- female mice was measured each week from 4-19 weeks of age. Data are presented as means ± SDs. * P<0.05. (H) The body weight of WT, PCBP4+/-, and PCBP4-/- female mice was measured each week from 20-45 weeks of age. (I) The body weight of WT, PCBP4+/-, and PCBP4-/- male mice was measured each week from 18-45 weeks of age. Figure S2 A B PCBP4-/- lung tissue PCBP4-/- lung tissue Figure S2. PCBP4 knockout mice are prone to lung cancer. (A-B) Representative images of lung tissues from two PCBP4-/- mice. The main tick mark of the ruler represents one centimeter. Figure S3 A Liver Spleen 100 µm 100 µm 50 µm 25 µm B Liver Kidney Pancreas Spleen 100 µm 100 µm 100 µm 100 µm 50 µm 50 µm 50 µm 25 µm Figure S3. PCBP4 knockout mice are prone to lymphoma. (A) Representative images of HE-stained sections of liver and spleen from PCBP4+/- mice. (B) Representative images of HE-stained sections of liver, kidney, pancreas, and spleen from PCBP4-/- mice. Figure S4 DAPI B220 CD3 50 µm 25 µm Figure S4. PCBP4 knockout mice are prone to B-cell lymphoma. Representative images of immunofluorescence-stained kidney sections of PCBP4-/- mice with antibodies against B-cell marker B220 and T-cell marker CD3, respectively. Figure S5 A B 100 µm 50 µm Figure S5. Kidney tumors in PCBP4-deficient mice. (A) Representative image of kidney tumor in one PCBP4-/- mouse. The main tick mark of the ruler represents one centimeter. (B) Representative images of HE-stained kidney tumor in one PCBP4+/- mouse. Figure S6 s y n u e r e t r g m n e e n i l a n y A v d a i p i e u h r N L S K H L T B L PCBP4 GAPDH 1 2 3 4 5 6 7 8 ) 6 B r e s l v e i l v 5 e n l i t s l p 4 i e r v c e s l n 3 a o t r t d 4 e 2 r P a B p C 1 m P o c ( 0 r t n y g s n e r N e e n u i a a v e n u L i l e m r d L L p i H y B S h K T Figure S6. The level of PCBP4 transcript in eight tissues of wild-type mouse. (A) RT-PCR was performed to measure the levels of PCBP4 transcript in mouse liver, spleen, kidney, heart, lung, thymus, brain, and lymph node (LN). GAPDH was measured and used as a loading control. (B) The relative levels of PCBP4 transcript measured in (A) were normalized by level of GAPDH and then compared to the level of PCBP4 in liver. Figure S7 A B A N 2.5 Ctrl CPT Dox R * * * m * 1 2.0 FL83B C2C12 7 8 P F l l T T x x 1.5 Z r r o o P P t t f D D C C C C o l 1.0 e p53 v e l 0.5 e 1 4.08 3.14 1 2.70 2.74 v i t a l 0.0 Actin e FL83B C2C12 R C D A N R 1.25 * C2C12 m 1 7 1.00 8 3 P 5 r F p c i siRNA) 0.75 Z s S f o p53 l e 0.50 v 1 0.15 e l 0.25 Actin e v i t a l 0 e Scr sip53 R Figure S7. The expression of ZFP871 is increased by activation of p53 but decreased by knockdown of p53 in cells. (A) The levels of p53 and actin proteins were measured in FL83B and C2C12 cells, untreated or treated with 300 nM camptothecin or 0.3 µg/ml doxorubicin for 24 h. (B) The level of ZFP871 mRNA was measured by qRT-PCR in FL83B and C2C12 cells, treated as in (A). Data were presented as means ± SDs normalized to actin mRNA from three separate experiments. *P<0.05. (C) The levels of p53 and actin proteins were measured in C2C12 cells transfected with scrambled siRNA or siRNA against p53 for 3 day. (D) The level of ZFP871 mRNA was measured by qRT-PCR in C2C12 cells, treated as in (C). Data were presented as means ± SDs normalized to actin mRNA from three separate experiments. *P<0.05. Figure S8 4-8-1 4-8-4 4-8-7 4-23-2 4-23-5 +/+ +/- -/- +/+ -/- PCBP4 PCBP4 p53 p21 Actin Mutation No a553c No a651c/a653c Deletion No 158-240 No Codon 1-40 C132W H165P/M166V Exon 2 5 5 Figure S8. The p53 gene is prone to mutation in immortalized PCBP4-deficient MEFs. The levels of PCBP4, p53, and p21 proteins were measured in two sets of immortalized WT, PCBP4+/- and PCBP4-/- littermate MEFs. Actin was used as a loading control. The mutation or deletion of the p53 gene was determined by sequencing p53 cDNA from each line of MEFs and listed below the western blots. Figure S9 1 2 - - 1 1 7 7 8 8 P P F F siRNA r Z Z c i i S s s (100nM,3d) p53 Actin Figure S9. The level of p53 transcript was measured in C2C12 cells transfected with a scrambled siRNA or one of the two unique siRNAs against ZFP871. Actin was measured and used as a loading control. Figure S10 A C2C12 B TS20 C Scr siRNA siZFP871 Scr siRNA siZFP871 L L E E N N U U T T ) 35 Scr % ( * s siZFP871 l 30 l e c * c 25 i I I t o P P t p A A 20 o p D D a f 15 o e g 10 a t n e 5 c r e P 0 d d e e C2C12 TS20 g g r r e e M M D C2C12 E TS20 F No treatment Dox No treatment Dox L L E E N N U U T T No treatment ) 50 * % ( Dox s l l * e 40 c c I I i t P P o t 30 A A p o D D p a f 20 o e g a t n 10 e c r e P d d 0 e e C2C12 TS20 g g r r e e M M Figure S10. Knockdown of ZFP871 and treatment with doxorubicin lead to apoptosis in C2C12 and TS20 cells. (A-B) The apoptotic cells were measured with TUNEL assay in C2C12 (A) and TS20 (B) cells transfected with scrambled siRNA or siRNA against ZFP871 for 3 d. (C) The percentage of apoptotic cells shown in (A-B). *P<0.05. (D-E) The apoptotic cells were measured with TUNEL assay in C2C12 (D) and TS20(D) cells untreated or treated with 0.3 µg/ml doxorubicin for 24 h. (F) The percentage of apoptotic cells shown in (D-E). *P<0.05..
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