Supplementary Methods
Cell culture, mice and reagents
Hypoxia-mimetic drugs DFX and CoCl2 (Sigma) were used at 200 µM, cycloheximide
(Sigma) at 25 µM, MG132 (Calbiochem) at 10 µM, rapamycin (Cell Signaling) at 25 nM.
For growth curves, rapamycin was added the day after seeding the cells and left
throughout duration of the experiment. Hypoxia was generated in In Vivo2 hypoxic
workstation (Ruskinn Technologies). For serum-starvation experiments, cells were
washed in PBS and incubated in 0.2% or no serum for the indicated time period.
For tumourigenesis assays, primary wt and Pml-/- MEFs were infected with pBabeRasV12 and pBabe-E1A retroviruses. Growth curves and soft agar transformation assays
were performed as described1. A few passages after selection (3 to 6 passages), 5 x 106
cells were injected subcutaneously into the right flank of 6-week-old athymic nude mice
(NCRNU-M, Taconic Farms, Inc.). For treatment with anti-VE-cadherin Ab, rat mAb
against mouse VE-cadherin (kindly provided by ImClone Systems) was diluted in PBS
and injected intraperitoneally at a dose of 20 mg per animal every 3 days, starting on the
day after tumour inoculation. Equal volume of vehicle was used as control.
VEGF-A quantization from cell supernatants or mouse plasma was performed using
the Quantikine Murine Immunoassay kit (R&D Systems) according to the manufacturer’s
instructions.
Hind-limb ischemia surgery
For ligation and segmental resection of left femoral vessels, a midline incision of
abdominal skin was made, and extra peritoneal dissection laterally along the tissue planes
was performed to expose the femoral vessels in left inguinal area. The artery was then
ligated both proximally and distally using 6-0 silk sutures and the ligated vessels were
resected between the ligatures without damaging the nervus femoralis. The midline
incision was then closed using 4-0 polysorb sutures. Rate of blood flow was measured
with a digital Laser Doppler flowmeter (Model ALF21, Advance Co.) every three days in
both the ischemic and non-ischemic hind-limbs of the same animal and ratios were
calculated.
Plasmids, cell transfection and transactivation assays
pCMV-Tag2B-PML-IV was described2. pCDNA3-HIF-1 contains full-length human
HIF-1 cDNA. HRE-luciferase (HRE-luc) contains three copies of the hypoxia response
element (HRE) of the phosphoglycerate kinase (PGK) gene cloned in pGL2 basic
(Promega).
For luciferase assays with Dual Luciferase Reporter Assay System (Promega), H1299
cells were transfected with 200 ng HRE-luc and 54, 107 and 252 ng PML, with or
without 20 ng HIF-1. Renilla expressing plasmid (10 ng) was cotransfected to normalize
for transfection efficiency. Luciferase activity was assayed 48 hours post transfection.
Primary MEFs were transfected with 500 ng HRE-luc and assayed as H1299 cells. MycRheb was from K. Guan. Transient transfections were performed with Effectene
transfection reagent (Qiagen). For co-IP experiments, HEK-293 cells were transfected
with 2 µg Myc-Rheb ± 3 µg Flag-PML. siRNA oligonucleotides for PML (5’AAGCACGAAGACAGACCTCTGG-3’ and 5’-AACGACAGCCCAGAAGAGGAA3’)3 and control oligos (Lamin A/C siRNA oligonucleotides purchased from Dharmacon)
were transfected with Lipofectamine 2000 (Invitrogen).
wt-SK6 and rapamycin-insensitive-S6K plasmids were a kind gift of G. Thomas.
Real-time PCR
Quantitative real-time PCR for Redd1 and hprt were performed on the Roche LightCycler
using light cycler DNA Master SYBR Green I (Roche). Primers sets were:
Redd1 Fwd (5’-CGAACTCCGGCCGCTGATCG-3’);
Redd1 Rev (5’-CAATCGCGCCTGGGACAGGC-3’);
hprt Fwd (5’-CCTGCTGGATTACATTAAAGCACT-3’);
hprt Rev (5’-GTCAAGGGCATATCCAACAACAAA-3’).
Western blotting
The following antibodies were used: anti-mouse Hif-1 (Novus Biologicals and Simon
laboratory); anti-human HIF-1 (BD Transduction Laboratories); anti-FLAG (Sigma);
anti-Myc, anti-P-S6K (T389), anti-S6K, anti-PS6 (S235/236), anti-S6, anti-P-4EBP1
(T37/46), anti-4EBP1, anti P-Akt (S473), anti-Akt, anti-mTOR (Cell Signaling); antiTsc2 (Santa Cruz); anti-human PML: PG-M3 (Santa Cruz) and rabbit anti-PML
(Chemicon), anti-mouse PML: S36 and S37 monoclonal antibodies (kindly provided by
Dr. S. Lowe); anti- -actin (Sigma); anti-Hsp90 (BD Transduction Laboratories); anti
laminB1 (AbCam); anti--tubulin (Sigma); anti-Rheb (. Anti-Redd1 was a gift from Dr.
J. Kaufmann.
TMA analysis
For analysis of human tumours, the study cohort was comprised of 99 primary and 51
metastatic prostate tumour biopsies. The patients were treated and followed at Memorial
Sloan-Kettering Cancer Center and tumour samples were collected at the time of surgical
resection with written informed consent. Tumour tissue microarray preparation, PML
(Santa Cruz) and CD34 staining (Dako) were performed as previously described4. PML
scoring system was based on counting PML-NBs in tumour cells as follows: no NBs=0;
few NBs (2 or fewer)=1; normal number of NBs (10-20)=2. Staining of endothelial cells
for CD34 was used to calculate microvessel density. CD34 positive clusters of cells were
counted as single microvessels, while larger vessels were not counted. Cases that had
more than 50% of the core composed of tumor cells were analyzed. The scoring system
we utilized is: 0-2 vessels=0; 2-5 vessels=1; >5 vessels=2 per 20 x field.
Statistical evaluation
Averaged data was analyzed for statistical significance by Student’s t-Test. Single nonaveraged values were compared separately for each time point. To compare PML and
CD34 staining scores, Pearson Chi-Square correlation was calculated with SPSS software
(version 11).
Supplementary References
1.
2.
3.
4.
Grisendi, S. et al. Role of nucleophosmin in embryonic development and
tumorigenesis. Nature 437, 147-53 (2005).
Bernardi, R. et al. PML regulates p53 stability by sequestering Mdm2 to the
nucleolus. Nat Cell Biol 6, 665-72 (2004).
Xu, Z. X., Timanova-Atanasova, A., Zhao, R. X. & Chang, K. S. PML colocalizes
with and stabilizes the DNA damage response protein TopBP1. Mol Cell Biol 23,
4247-56 (2003).
Gurrieri, C. et al. Loss of the tumor suppressor PML in human cancers of multiple
histologic origins. J Natl Cancer Inst 96, 269-79 (2004).
Supplementary Figure Legends
Supplementary Figure 1 | PML controls Hif-1 activity and accumulation rate in
hypoxia. a-b, Hif-1 transactivation assays. (a) H1299 cells transfected with PML and
treated with hypoxia for 8 hours. (b) wt and Pml-/- MEFs transfected with HRE-luciferase
and treated with hypoxia as indicated. Results are presented as mean ± s.d. from one
experiment performed in triplicate. c, VEGF concentration in media from wt and Pml-/MEFs treated with hypoxia. Results are presented as mean ± s.d. from three independent
experiments. d, Western blot of Glut-1 in wt and Pml-/- MEFs treated with hypoxia. e,
Western blot of Hif-1 in wt and Pml-/- MEFs treated with hypoxia and CoCl2 as
indicated. Numbers express Hif-1 levels upon normalization for hsp90, expressed as
fold induction over control. f, Western blot of Hif-1 in wt and Pml-/- MEFs treated with
hypoxia for 6 hours followed by treatment with MG132. Hif-1 levels are expressed as in
e. All experiments were repeated at least three times with similar results.
Supplementary Figure 2 | PML negatively regulates HIF-1 accumulation in a
pVHL-independent manner. a, Western blot of HIF-1 and PML in MCF7 cells stably
transfected with empty vector or PML upon treatment with DFX or CoCl2 for 6 hours. b,
Western blot of HIF-1 and PML in SW-620 cells stably transfected with empty vector
or PML upon treatment with DFX or CoCl2 for 6 hours. c, Western blot of HIF-1 and
PML in PC3 cells stably transfected with empty vector or PML upon treatment with
hypoxia. d, Western blot of HIF-1 and PML in PC3 cells stably transfected with empty
vector or PML upon treatment with hypoxia for 4 hours and treatment with CHX and
MG132 as indicated. HIF-1 levels in a-d are expressed as fold induction over control
upon normalization for actin. e, Western blot of HIF-1 and Flag-PML in RCC-4 cells
transiently transfected with increasing amounts of PML and treated with MG132 for 4
hours where indicated. All experiments were repeated at least three times with similar
results.
Supplementary Figure 3 | mTOR inhibition is defective in Pml-/- MEFs in hypoxia
and serum starvation. a, Western blot of Hif-1 in wt and Pml-/- MEFs treated with
hypoxia as indicated and co-treated with rapamycin for 12 hours. Hif-1 levels are
expressed as fold induction over control upon normalization for hsp90. b, Western blot
analysis of P-S6K and total S6K in wt and Pml-/- MEFs treated with CoCl2. c, Western
blot analysis of P-S6K and total S6K in wt and Pml-/- MEFs treated with 0.2% serum. d,
Western blot analysis of P-S6 and total S6 in 293 cells upon control and PML siRNA
transfection and incubation in 0% serum for 48 hours. e, Western blot of P-Akt and total
Akt in wt and Pml-/- MEFs treated with DFX. Bar graphs in b-e represent phosphorylation
levels upon normalization for total protein levels and  -actin. f, Real-time PCR for Redd1
in wt and Pml-/- MEFs treated as indicated. Bars represent relative amount of Redd1
mRNA upon normalization for hprt mRNA. g, Western blot analysis of Tsc2, PML, HIF1 and Redd1 in PC3 cells transfected with control and PML siRNA 48 hours posttransfection. All experiments were repeated at least three times with similar results.
Supplementary Figure 4 | PML inhibits mTOR activity and colocalizes with it in the
nucleus upon hypoxia. a, Western blot analysis of P-S6K, S6K, P-S6, S6, P-4EBP1 and
total 4EBP1 in HEK293 cells transfected with increasing amounts of PML. Numbers
below panels represent phosphorylation levels upon normalization for total protein and
actin. b, Western blot analysis of P-S6K, S6K, P-S6 and S6 in 293 cells transfected with
wt or rapa-insensitive S6K and co-transfected with PML. Cells were treated with 0.1%
serum 24 hours before harvesting. c, Western blot analysis of PML, P-S6, S6, Rheb and
actin in 293 cells transfected with Flag-PML and Myc-Rheb and treated with hypoxia.
Bar graphs represent phosphorylation levels upon normalization for total protein levels
and -actin d, IP with anti-Myc antibody from 293 cells transfected with Myc-Rheb with
or without Flag-PML and treated with 0.2% O2. Panels represent levels of Rheb and
mTOR in IP. Bar graph represents mTOR and Rheb ratios in IP as mean of three
independent experiments ± s.d. e, Subcellular localization of mTOR, PML and Rheb in
HEK-293 treated with DFX. LaminB and -tubulin are used as controls. f, Representative
confocal images of immunofluorescence staining of Pml and mTOR (Upstate Ab) in wt
and Pml-/- MEFs upon treatment with 0.1% O2. All experiments were repeated at least
three times with similar results.
Supplementary Figure 5 | Properties of transformed MEFs and expression of PML
in human prostate tumours. a, Growth curve of wt and Pml-/- MEFs transformed by
infection with RasV12 and E1A oncogenes. Results are presented as mean ± s.d. from one
experiment performed in triplicate. b, Number of colonies formed in soft agar by wt and
Pml-/- MEFs transformed with RasV12 and E1A. Results are presented as mean ± s.d. from
one experiment performed in triplicate. c, Western blot showing expression levels of
RasV12 and E1A in two sets of wt and Pml-/- transformed MEFs. d, H&E staining of wt
and Pml-/- tumours. Indents show areas of hemorrhage and graph represents average
number of big hemorrhagic lesions, defined as enlarged vascular lesions, per 10x field. e,
f, Quantification of CD31-positive microvessels (e) and P-H3-positive cells (f) per 40x
field at the indicated times upon injection. Graphs in d-f: 10 fields per tumour were
counted. Results are presented as mean ± s.d. g, Tumour growth in nude mice injected
with wt (left) and Pml-/- (right) transformed MEFs and treated with VE-cadherin mAb.
Results are presented as mean ± s.d. h, Correlation between PML and CD34 expression
with tumour grade in CaP. i, Example of PML and CD34 immunostaining in a metastatic
human prostate tumour. Arrows point to PML-positive endothelial cells in the upper
panel and blood vessels in the lower panels.