Cell, Volume 128

Supplemental Data

PRAK Is Essential for ras-Induced

Senescence and Tumor Suppression Peiqing Sun, Naoto Yoshizuka, Liguo New, Bettina A. Moser, Yilei Li, Rong Liao, Changchuan Xie, Jianming Chen, Qingdong Deng, Maria Yamout, Meng-Qiu Dong, Costas G. Frangou, John R. Yates III, Peter E. Wright, and Jiahuai Han Supplemental Experimental Procedures Generation of PRAK Knockout Mice Genomic DNA clones covering the PRAK coding region was isolated from a 129/sv strain mouse genomic DNA library and used to construct the targeting vector (Figure S1). The targeting vector was transfected into R1 ES cells, and the G418 and gancyclovir double resistant colonies were isolated and expanded. Genomic DNA was purified and screened for homologous recombination by Southern blotting with the 3' external probe. Two independent ES clones were injected into blastocysts of C57BL/6J mice. Male mice with a high degree of chimerism were crossed to C57BL/6J females to generate PRAK+/− mice. DNA was prepared from the tails of 2-week-old progenies. Genotyping was performed by Southern blot analysis or by PCR analysis of tail- and embryo-derived DNA. Primers A (5’-gagatgttcatgctgctgctgca-3’) and B (5’-ccaaagagcaagcacacattaaa-3’) were used to detect the wild type allele with a PCR product of 500bp. Primers A’ (5’-gcgaggccagaggccacttgtgta-3’) and B’ (5’-ccaaagagcaagcacacattaaa-3’) were used to detect the PRAK KO allele with a PCR product of 700bp. PCR was performed using these primers by 1 cycle at 95°C for 5 min followed by 35 cycles at 94°C for 30 s, 62°C for 30 s, 72°C for 1 min, with an extension step of 7 min at 72°C at the end of the last cycle. Analysis of Cell-Cycle Profiles BJ cells either treated with DNA-damaging agents or transduced with ras were grown in fresh complete medium for overnight before labeled with 30 µM BrdU (Sigma) for 1-2 hours. Cells were collected by trypsinization and fixed in 70% ethanol at 4 C for overnight. Cells were then treated with 2 N HCl/0.5% Triton X-100 for 30 min at room temperature, followed by neutralization with 0.1 M Na2B4O7 and subsequent incubation with FITC-conjugated anti-BrdU antibody (Becton-Dickinson) for 1 hour at room temperature. Finally, cells were washed with PBS/1%BSA/0.5% Tween-20, resuspended in PBS containing 5 µg/ml propidium iodide and analyzed by two-dimensional flow cytometry to detect both fluorescein and propidium iodide. Analysis of Eµ-N-rasG12D Mice Eµ-N-rasG12D transgenic mice (a gift from Dr. A.W. Harris) were crossed with PRAK+/- mice, and mice with Eµ-N-ras/PRAK+/+, Eµ-N-ras/PRAK+/- and Eµ-N-ras/PRAK-/- were selected and observed for lymphoma free survival.

Figure S1. Generation of PRAK Knockout Mice (A) A targeting vector was made to replace exon 8 of the PRAK gene. Neo and TK genes in the vector were driven by PGK promoters in an antisense orientation of the PRAK gene. A self-cleaving ribozyme sequence (Rib), which has been shown to result in ~100% self-cleavage of mRNA containing this sequence (Zarubin T, Cell Res.), was included in front of PGK promoter of Neo in the sense orientation of the PRAK gene. This ribozyme destroys all the possible fusion mRNA driven by the PRAK promoter after homologous recombination. The position of the probe used in the Southern blot analysis is indicted. The position of the primers used for genotyping are indicted as A, B, A’, and B’. H and X indicate HindIII and XbaI sites, respectively.

(B) The sequence of the ribozyme used in the targeting vector.

(C) Genomic DNA from tails of the mice with indicated genotypes was digested by HindIII and XbaI, and analyzed by Southern blotting, using the probe indicated in A. The 9 kb and 6 kb bands were derived from the wild type and mutant alleles, respectively.

(D) PCR-based genotyping detected wild type genome with a product of 500 bp using the A-B primer set, and PRAK KO genome with a product of 700 bp using the A’-B’ primer set.

(E) Total RNA was isolated from MEF cells or liver tissues of wild type or PRAK KO mice, and analyzed by Northern blot using randomly primed PRAK cDNA as probe (top panel). Ethidium bromide-staining of 28s and 18s rRNA indicates equal loading of RNA (middle panel). The same RNA samples were also analyzed by RT-PCR using a primer set amplifying the first 250 bp of the PRAK coding region (bottom panel). The mRNA of PRAK, either full length or truncated forms, was undetectable in PRAK KO mice due to ribozyme-mediated mRNA degradation.

(F) MEF cells from mice with indicated genotypes were analyzed by Western blot analysis to detect PRAK and actin.

Figure S2. PRAK Deficiency Does Not Promote Tumor Induction by DMBA and TPA in a Two-Stage Protocol

(A) PRAK+/+, PRAK+/- and PRAK-/- littermates were treated with a tumor initiator DMBA, and then with a tumor promoter TPA twice a week starting 1 week after the initial DMBA treatment. Days of TPA promotion before tumors were visible is shown for each genotype. Values are mean ± standard deviations (SD). p > 0.05 for both PRAK+/- vs PRAK+/+ and PRAK-/- vs PRAK+/+ in Student’s t-Tests.

(B) Expression of SA-β-gal and immunohistochemical analysis of indicated protein levels in papillomas induced by DMBA+TPA in mice of specified genotypes. Arrows indicate areas of SA-β-gal positive cells. Squares indicate areas shown at the 40X magnification.

Figure S3.

(A) SA-β-gal expression in additional sections of DMBA-induced wild type skin papilloma. Frozen 10 µm sections of DMBA-induced wild type skin papilloma were stained for SA-β-gal and then counter stained with eosin.

(B) Contiguous sections of DMBA-induced wild type skin papilloma were stained for hematoxylin and eosin, SA-β-gal and p16INK4A. The boxes indicate the corresponding regions of the same squamous epithelium in different sections.

(C) PRAK deficiency does not alter the expression of SA-β-gal marker in aged mouse skins. Frozen 10 µm sections of normal skins from a newborn (15-day old) wild type (WT) mouse or adult (14-month old) wild type (WT), PRAK+/- (HT) or PRAK-/- (KO) mice, and the normal skin adjacent to a DMBA-induced papilloma in an adult PRAK deficient mouse were stained for SA-β-gal and counter stained with eosin.

Figure S4. PRAK Deficiency Prevents Induction of Senescence Markers in DMBA-Induced Skin Papillomas Comparison of indicated protein levels in DMBA-induced PRAK+/+ (WT) and PRAK+/- (HT) papillomas and adjacent normal skins by Western blot. * denotes a nonspecific band.

Figure S5. Expression of SA-β-Gal and Other Senescence Markers during ras-Induced Senescence in MEFs and BJ Cells (A) Representative fields of wild type (WT), PRAK+/- (PRAK HT), PRAK-/- (PRAK KO) and p53-/- (p53 KO) MEFs stained for SA-β-gal on day 7 post transduction with Ha-RasV12 or vector control. (B) Representative fields of BJ cells expressing shRNA for GFP or PRAK, stained for SA-β-gal on day 8 post transduction with Ha-RasV12 or vector control. (C) Western blot analysis indicated protein levels in wild type (WT), PRAK+/- (HT), and PRAK-/- (KO) MEFs on day 8 post transduction with Ha-RasV12 or vector control. (D) Western blot analysis indicated protein levels in BJ cells expressing shRNA for GFP (shGFP) or PRAK (shPK-1, shPK-2 or both shPK-1 and -2) on day 8 post transduction with Ha-RasV12 or vector control.

Figure S6. PRAK Is Essential for ras-Induced Senescence in BJ Cells at Early Passages (A) Population doubling of BJ cells transduced with shRNA for GFP or PRAK and Ha-RasV12 or vector control were followed over 10 days, starting at PD15. Values are mean ± standard deviations (SD) for duplicates.

(B) Representative fields of the same BJ cell populations stained for SA-β-gal on day 9 post transduction with ras or vector.

(C) Percentage of SA-β-gal positive cells in the same BJ populations on day 4,7 and 10 post transduction with ras.

(D) Western blot analysis showing knockdown of PRAK protein level by PRAK shRNA in BJ cells.

(E) Population doubling of BJ cells transduced with shRNA for PRAK (shPK-1 or -2) or scrambled shPK-1 sequence and Ha-RasV12 or vector control were followed over 14 days, starting at PD30. Values are mean ± standard deviations (SD) for duplicates.

(F) Percentage of SA-β-gal positive cells in the same BJ populations as in (E) on day 5 and 8 post transduction with ras.

(G) Western blot analysis showing knockdown of PRAK protein level by PRAK shRNA in BJ cells transduced with shRNA for PRAK (shPK-1 or -2) or scrambled shPK-1 sequence.

Figure S7. PRAK Is Not Essential for DNA-Damage-Induced G1 Arrest and p53 Transcriptional Activity

(A) BJ cells transduced with pSUPER.retro-shPRAK, -shp53 or -shGFP were either left untreated or treated with 5 Gy γ-irradiation or 50 J/m2 UV at PD27. After recovery in complete medium for 15 hrs, cells were labeled with 30 µM of BrdU. Cells were then fixed, stained with a FITC-conjugated anti-BrdU antibody and propidium iodide and analyzed by two-dimensional flow cytometry to detect both fluorescein and propidium iodide. The percentage of BrdU-positive (S-phase) cells is indicated for each cell population.

(B) BJ cells with a stably integrated p53-dependent luciferase reporter (PG-Luc) or a non-p53-dependent control reporter (MG-Luc) were transduced with pSUPER.retro-shPRAK, -shp53 or -shGFP. Cells were either left untreated or treated with 30 J/m2 UV at PD32. Luciferase activity was measured after recovery in complete medium for 22 hrs and normalized to protein concentrations. Values are mean ± standard deviations (SD) for triplicates.

(C) Western blot analysis showing levels of indicated proteins in BJ cells at PD30 transduced with shRNA for GFP or PRAK, 20 hrs after treatment with 50 J/m2 UV (UV) or left untreated (Con).

Figure S8. Activated ras and MKK3 Induces the Kinase Activity of Ectopically Expressed PRAK during Senescence in BJ Cells BJ cells were transduced with a HA-tagged PRAK or its vector control (BabePuro) and Ha-RasV12, MKK3E or their vector control (WZLHygro) at PD32.5. PRAK was immunoprecipitated from these cells on day 6 post ras transduction using an anti-HA antibody, and assayed for kinase activity toward HSP27 in the presence of [γ-32P]ATP. Western blot analysis was performed to examine the PRAK level in the immunoprecipitates and the levels of PRAK and actin in cell lysates.

Figure S9. PRAK Overexpression Enhances ras-Induced Increase in Cell Volume but Does Not Promote Apoptosis (A) BJ cells transduced with HA-tagged wild type (WT) or mutant (KM or 182A) PRAK or their vector control (BabePuro) (VT) and Ha-RasV12 or its vector control (WZLHygro) (VT) were measured for cell volume using a CASY cell counter (Schärfe System GmbH) at PD29 on day 9 post ras transduction. Values are mean ± standard deviations (SD) for duplicates.

(B) The same BJ cell populations at PD34 on day 6 post ras transduction were labeled with 50 mM BrdU for 1 hr, harvested, stained with a FITC-conjugated anti-BrdU antibody and propidium iodide and analyzed for BrdU incorporation and DNA contents by two-color flow cytometry. The percentage of cells with a <1N (sub-G1) DNA content is shown for each population.

Figure S10. p38 Phosphorylates p53 at Ser33 upon ras Activation (A) p38 immunoprecipitated from BJ cells at PD29 on day 7 post transduction with Ha-RasV12 or vector control was assayed for kinase activity toward ATF2, wild type p53(1-61) or indicated mutants of p53(1-61). Relative kinase activity and Coomassie Brilliant Blue R-stained substrate input are shown. Western blot was performed to detect indicated protein levels in immunoprecipitates and lysates.

(B) Phosphorylation of wild type p53(1-61) or indicated mutants of p53(1-61) by recombinant p38 in vitro in the presence of [γ-32P]ATP. Relative kinase activity and Coomassie Brilliant Blue R-stained substrate input are shown.

(C) Western blot analysis showing levels of p53 phosphorylated at Ser15 or Ser33, total p53 and actin in BJ cells at PD25 on day 8 post transduction of Ha-RasV12 or vector control.

Figure S11. Ser37, Ser15, and Ser33 Are Required for p53 to Mediate ras-Induced Senescence (A) Western blot detecting indicated proteins in BJ cells transduced with a binary vector (GAPDH-PGK-Puro) (VT) for wild type or mutant hp53 or BabePuro-hp53 (BP-p53) (top), or with the binary p53 constructs and Ha-RasV12 or vector (bottom). Endo-p53, endogenous p53; Exo-p53, ectopically expressed p53.

(B) Population doublings of BJ cells transduced with wild type or mutant hp53 or vector (GAPDH-PGK-Puro) (VT) and Ha-RasV12 or vector (VT) were followed over 7 days, starting at PD35. Values are mean ± standard deviations (SD) for duplicates.

Figure S12. Ectopic Expression of p53 at Low Levels from the Binary Vector Did Not Promote Apoptosis in BJ Cells in the Presence or Absence of Oncogenic ras BJ cells transduced with wild type (WT) or mutant (S15A, S33A or S37A) hp53 or their vector control (GAPDH-PGK-Puro) (VT) at PD31 on day 4 post ras transduction were labeled with 50 mM BrdU for 1 hr, harvested, stained with a FITC-conjugated anti-BrdU antibody and propidium iodide and analyzed for BrdU incorporation and DNA contents by two-color flow cytometry. The percentage of cells with a <1N (sub-G1) DNA content is shown for each population.