Nature manuscript 2004-12-28075C
Supplementary Figure Legends
Supplementary Figure S1. Scheme of Pten and Trp53 conditional knockout alleles. a,
Murine Pten and (b) Trp53 loci, before and after Cre-mediated recombination with
location of loxP (triangles) and PCR-primer sites (P1, P2 and P3). c, PCR-based
identification of mutants from various non-recombined allelic combinations of PtenloxP
and Trp53loxP alleles prior to Cre-mediated recombination on tail DNA.
Supplementary Figure S2. Specificity of recombination. a, Recombination of PtenloxP
and Trp53loxP alleles is restricted to the anterior prostate (AP), ventral prostate (VP),
dorsolateral prostate (DLP), and occurs to a minor degree in the seminal vesicles (SV).
LI, large intestine. PCR analysis was performed on DNA extracted from 9-week-old
mice. b, H&E staining of VP and DLP at 9 weeks. Arrows indicate sites of local
invasion. c, H&E staining of AP at 6 months. d, Immunohistochemical (IHC) staining
with Androgen Receptor (AR) (glandular epithelial marker) and p63 (basal cell marker)
antibodies at 11 weeks reveals no obvious differences in localization or expression of
these epithelial cell markers upon p53 loss. Scale bars, 50 µm.
Supplementary Figure S3. In vitro modelling of acute loss of Pten in MEFs. a, Pten and
phospho-Akt status in MEFs after Cre-mediated recombination via MSCV-Cre. b, Upper
panel: growth curves of primary MEFs, infected with Adenovirus Cre (no selection) and
followed over a 6 day period: Pten heterozygous (Pten/+-Ad-Cre: squares), null (PtenAd-Cre: triangles), and wt cells (Pten+/+-Ad-Cre: circles). Lower panel: cellular
senescence assay of cells from upper panel. c, MEFs after Cre-mediated recombination
of loxP alleles in Pten via Ad -Cre. d, Western blotting shows the Pten knockdown via
RNA interference in MEFs. Lane 1 -Pten wt infected with empty vector; Lane 2 -Pten
wt infected with Pten shRNA; Lane 3 -Pten+/- infected with empty vector; Lane 4 -Pten+/infected with random shRNA; Lane 5 -Pten+/- infected with Pten shRNA. e, Pten and
Trp53 synergize in a MEF based cell proliferation assay. Triplicate mean values for cell
numbers after 6 days in culture are shown. Error bars indicate s.d. f, p53 induction upon
acute Pten loss in MEFs is not due to p53 phosphorylation at serine 15 site. Ctrl, no virus;
Vec, MSCV-empty vector; L/L, Pten loxP/loxP. g, Western blotting of MEFs reveals that
induction of p53 induction and Plasminogen Activator Inhibitor-1 (PAI-1) upon loss of
Pten.
Supplementary Figure S4. a, In vivo status of proliferation and signalling markers in the
prostates of the indicated mutants and wt mice. Immunohistochemical (IHC) analysis of
Pten, phospho-Akt (pAkt), and Ki-67 in anterior prostates at 11 weeks. b, double staining
of -gal (red arrows) and Ki-67 (black arrows) in Pten-deficient versus Pten-Trp53
double mutant prostates.
Supplementary Figure S5. In vitro senescence analysis. Senescence, as measured by gal activity in MEFs of indicated genotypes after infected with MSCV-Cre (a), Pten
shRNA (b) or Ad-Cre (d). c, Infection of wt MEFs with pBabe vector or pBabe
expressing mAkt. Bar, 100 µm.
Supplementary Figure S6. a, AP lobes of indicated genotypes reveal necessity for
complete Pten loss for induction of senescence as measured by -gal activity. Bars,
2
50µm. b, low magnifications of AP stained for -gal activity from wt, Pten null and
Pten-Trp53 double null mice. Bar, 10µm.
Supplementary Table S1. Cryosections prepared from twelve different human radical
prostatectomy specimens (collected under IRB approval) were analyzed for senescence
with -gal staining. Each individual specimen typically was somewhat heterogeneous,
and included some regions of frank carcinoma, some regions of prostatic intraepithelial
neoplasia (PIN), and occasionally small regions of relatively normal-appearing glands. A
small subset of glands within these tissues, particularly with the histologic characteristics
of hyperplasia/PIN, showed strong -gal staining. In the scoring system used here, +++
and ++ indicated strong ß-gal staining, easily detectable at 100x magnification; +
indicated weak ß-gal staining, only detectable at 400x magnification; * denotes Patient B,
** denotes Patient A in Figure 4f.
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