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Supplementary Figure Legends
Supplementary figure 1. Senescence-associated-ß-galactosidase (SA-ß-gal) activity of
confluent WI-38hTERT/GFP-RAF1-ER cells (left panel) and WI-38hTERT/GFP-RAF1-ER
cells induced into senescence by incubation with 20 nM 4-HT for 3 days (right panel). Cells
were stained for SA-ß-gal as described (Debacq-Chainiaux et al., 2009), and then with DAPI
to visualize nuclear DNA with a fluorescence microscope. The product produced by SA-ß-gal
cleavage of the 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) substrate was
observed in phase bright illumination as a black precipitate. The images shown are overlays
of the DAPI and the phase bright images. Approximately 65% of cells stained positively for
SA-ß-gal activity after 3 days of incubation with 4-HT although greater than 95% of cells
were senescent in terms of the presence of SAHFs and the inability to incorporate BrdU.
Approximately 5% of confluent control cells also stain positively. Note that the protocol used
to assay SA-ß-gal activity perturbs the structure of the SAHFs.
Supplementary figure 2. Proliferating WI-38hTERT/GFP-RAF1-ER cells that stain
positively for H2AX also stain positively for the S phase marker PCNA. For these
experiments, cells were pre-extracted with PBS containing 0.1% Triton-X-100 for 30 seconds
before formaldehyde fixation in order to remove PCNA that was not stably associated with
chromatin. This protocol enhances the visualization of PCNA in S-phase cells.
Supplementary figure 3. Treatment of WI-38hTERT/GFP-RAF1-ER or IMR-90/GFPRAF1-ER cells with 200 nM UCN-01, 10 µM KU-55933, or 10 mM N-acetyl-cysteine
(NAC) does not inhibit senescence induced by incubation of these cells with 20 nM 4-HT for
three days. (a) WI-38hTERT/GFP-RAF1-ER cells or (c) IMR-90/GFP-RAF1-ER cells were
incubated in the presence or the absence of 20 nM 4-HT with the indicated molecules for
three days. Fresh medium was then added containing 50 µM BrdU for 1 day or 4 days and the
percentage of cells incorporating BrdU was determined. (b) Representative microscopic
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images of WI-38hTERT/GFP-RAF1-ER cells incubated in the presence or absence of 4-HT
and the indicated molecules for three days. Cells were then fixed and analyzed by
immunofluorescence for BrdU incorporation, and by DAPI staining of nuclear DNA to
observe the presence or absence of SAHFs. (d) DAPI staining of nuclear DNA to visualize
SAHFs in IMR-90/GFP-RAF1-ER cells treated with 4-HT or 4-HT and the indicated
molecules for three days.
Supplementary figure 4. Only cells that traverse S phase show signs of DNA damage during
4-HT-induced senescence of IMR-90/GFP-RAF1-ER cells. (a) Shown are the percentage of
cells staining positively for H2AX foci, or p21, or phospho-T68-Chk2 by
immunofluorescence analyses. Control represents proliferating IMR-90/GFP-RAF1-ER cells.
4HT indicates cells treated with 20 nM 4-HT for three days. 4-HT + UCN-01 indicates cells
treated with 20 nM 4HT + 200 nM UCN-01 (Sigma U6508) for three days. 4HT+ KU-55933
indicates cells treated with 20 nM 4-HT+ 10 µM KU-55933 (Calbiochem 118502) for three
days. 4HT + NAC indicates cells treated with 20 nM 4-HT+ 10 mM N-acetyl-cysteine (NAC)
for three days. (b) Representative images of immunostaining of H2AX, p21, and phosphoT68-Chk2 as well as DAPI staining of nuclear DNA for cells induced in senescence by
treatment with 20 nM 4-HT for three days. (c) Cells were incubated with 50 µM BrdU for
three days in the absence (Control) or the presence of 20 nM 4-HT and, where indicated, 200
nM UCN-01, or 10 µM KU-55933, or 10 mM NAC. Shown is the percentage of cells having
incorporated BrdU, or staining positively for H2AX, or staining positively for both BrdU and
H2AX. (d) Representative images of immunostaining for BrdU and H2AX as well as DAPI
staining of nuclear DNA for cells induced in senescence by treatment with 20 nM 4-HT for
three days. The histograms in (a) and (c) show the means and standard deviations for three
independent experiments.
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Supplementary figure 5. Flow cytometry profiles to determine relative ROS levels. The
fluorescent ROS sensor dichlorodihydrofluorescein diacetate (H2DCFDA) was used to
determine ROS levels in living cells by flow cytometry as described in the Materials and
Methods. Data from a FACS Calibur were imported into R (Team, 2009) using the flowCore
package (Hahne et al., 2009) for statistical analyses. Shown are graphs for each culture in
which the X-axis displays the distributions of Log10 values for the fluorescence emitted by
individual cells (FL1-H) and the Y-axis displays the number of cells with a given Log10
(FL1-H) value after normalizing the peak number of cells to 1. The FL1-H value of
proliferating cells in 5% oxygen was set to 100.
Above each graph is indicated the
experimental condition in terms of oxygen concentration. All cells were incubated for three
days before flow cytometric analyses. Where indicated, 4HT was added to 20 nM and NAC to
10 mM. H2O2 refers to treatment of cells with 10 µM hydrogen peroxide for 10 minutes
directly before flow cytometry. The mode for the 5% O2 experimental condition of each
replicate is displayed as a black vertical bar in all panels.
Supplementary figure 6. Absolute quantification of CKIs in 50 µgs of whole cell protein
extracts of WI-38hTERT/GFP-RAF1-ER cells induced into senescence by treatment with 20
nM 4-HT for the indicated number of days. Purified recombinant proteins at the indicated
amounts were loaded on the same gel as standards and the membranes were scanned on an
Odyssey infrared image scanner after immunoblotting with antibodies to the indicated CKIs.
GST-p16, GST-p21, and p27-6His were purchased from Santa Cruz Biotechnology, whereas
GST-p15 was purchased from Abnova (Taiwan). To estimate the average abundance of p16 in
senescent cell extracts, we counted the number of cells used to prepare whole cell extracts. 50
µgs of senescent cell extract derived from approximately 120,000 cells contained an amount
of p16 giving an immunoblot signal equivalent to that obtained with 2 ng of GST-p16
corresponding to about 5 x 10-14 moles or 3 x 1010 molecules for 120,000 cells. Thus,
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senescent cells are estimated to contain on average approximately 250,000 molecules of
p16/cell, athough immunofluorescence analyses showed a highly heterogeneous expression of
p16 at the single cell level (Figure 5b).
Supplementary figure 7. Short-term proliferation assays to test the effects of CKI knockdown on establishment and maintenance of RAF-induced senescence. Shown are the mean
and standard deviation in cell number increases for 3 replicate experiments. (a) Effect of CKI
knock-down on establishement of RAF-induced senescence. WI-38hTERT/GFP-RAF1-ER
cells were treated with the indicated siRNAs for 24 hours and then incubated with medium
containing 20 nM 4-HT for three days. The increase in cell number is shown after subtraction
of the value for the si No Target. (b) Effect of CKI knock-down on maintenance of RAFinduced senescence. WI-38hTERT/GFP-RAF1-ER cells were induced into senescence by
incubation with medium containing 20 nM 4-HT for 3 days. Cells were then incubated with
fresh medium in the absence of 4-HT for three days followed by a treatment with the
indicated siRNAs for 48 hours. Cells were then incubated with fresh medium for 3 days. The
increase in cell number is shown after subtraction of the value for the si No Target control.
Method: 5000 cells were seeded in 96 well plates in triplicate for each experimental
condition. After the experimental incubations described above, cells were fixed with PBS +
2% formaldehyde for 10 minutes, and then nuclear DNA was stained with PBS + 5 µg/ml
Hoechst 33342 for 1 hour. After washing with PBS, Hoechst 33342 fluorescence was
measured using a UV microplate reader. This UV value was then used to calculate cell
numbers by comparison with a standard curve obtained by determining the fluorescence
values associated with known quantities of WI-38hTERT/GFP-RAF1-ER cells seeded in the
same microwell plates.
Supplementary figure 8. Reversion of RAF-induced senescence is accompanied by apparent
mitotic catastrophes in a significant minority of cells. Senescent WI-38hTERT/GFP-RAF1-
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ER were obtained by incubation with 20 nM 4-HT for 3 days. Reversion was induced by
withdrawing 4-HT and treating the cells with siRNAs directed against p16-INK4A and p21CIP1. Four days later, cells were fixed with formaldehyde and nuclear DNA was stained with
DAPI. SAHFs were lost in most cells and approximately 10% of cells showed aberrant
nuclear morphologies (arrows) suggesting cell death. The focus is on the micronuclei that are
in a different plane from the other nuclei in this image. Such aberrant nuclear structures were
observed in less than 1% of cells treated with No Target or RAF1 siRNAs.
Supplementary figure 9. Efficient reversal of RAF-induced senescence by simultaneous
siRNA knock-down of p16-INK4A and p21-CIP1 in WI-38hTERT/GFP-RAF-ER or IMR90/GFP-RAF-ER cells maintained in senescence by incubation with 20 nM 4-HT for 3, 5, or
7 days as indicated. No Target siRNAs were used as a negative control. 4 days after
transfection, cells were incubated with 50µM BrdU for 24hrs before anti-BrdU
immunofluorescence. Note that a small fraction of cells (approximately 2%) in these
populations does not express the GFP-RAF1-ER fusion protein and does not undergo
senescence in the presence of 4-HT. The continued proliferation of these cells in the presence
of 4-HT leads to an increase with time of the background signal of BrdU incorporation due to
cells that did not undergo senescence.
Debacq-Chainiaux F, Erusalimsky JD, Campisi J, Toussaint O (2009). Protocols to detect
senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells
in culture and in vivo. Nat Protoc 4: 1798-806.
Hahne F, LeMeur N, Brinkman RR, Ellis B, Haaland P, Sarkar D et al (2009). flowCore: a
Bioconductor package for high throughput flow cytometry. BMC Bioinformatics 10: 106.
R Development Core Team (2009). R: A language and environment for statistical computing.
R Foundation for Statistical Computing: Vienna, Austria. ISBN 3-900051-07-0.
http://www.R-project.org.
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