CDD-11-0226v2
Faraonio et al.
SUPPLEMENTARY INFORMATION
A set of miRNAs participates to the cellular senescence program in
human diploid fibroblasts
Supplementary Figure 1
Supplementary Figure 1  Characterization of senescent IMR90 cells.
Primary human fibroblasts IMR90 were grown in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% (v/v) fetal bovine serum and 1%
penicillin/streptomycin (Invitrogen). The population doubling level (PDL) was
calculated by using the formula: ΔPDL = log(nh/ni)/log2, where ni is the initial
number of cells and nh is the final number of cells at each passage. The cells
were used at 33 PDL or 58 PDL; at PDL58 most cells showed a senescent
phenotype as indicated by senescence-associated β-galactosidase (SA-β-gal)
staining and gene expression profile. (a) Representative images showing SA-β-
CDD-11-0226v2
Faraonio et al.
SUPPLEMENTARY INFORMATION
gal stain in young (PDL 33) and senescent (PDL 58) IMR90 cells. At least 300
cells were counted. (b) Western blot analysis of cyclin-dependent kinase (CDK)
inhibitors p21WAF1 and p16INK4A in whole-cell lysates prepared from young
(PDL 33) and senescent (PDL 58) IMR90 cells. GAPDH levels served as a
loading control. (c) Quantitative real-time PCR (Q-RT-PCR) analysis of cyclin A,
thymidylate synthase (TYMS), cyclin-selective ubiquitin carrier protein (UBE2C),
interleukin-6 (IL6) and forkhead box M1 (FOXM1) mRNAs in senescent versus
young IMR90 cells. Results are the mean of a triplicate determination.
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CDD-11-0226v2
Faraonio et al.
SUPPLEMENTARY INFORMATION
Supplementary Figure 2
Supplementary Figure 2  Characterization of cellular senescence induced
by DEM or etoposide treatment. (a) Young (PDL 33) IMR90 cells were
chronically exposed to diethylmaleate (DEM) 100 µM for 10 days before
harvesting. Cellular senescence was verified by SA-β-gal staining. At least 300
cells were counted. Representative images of control and DEM-treated cells are
reported. (b) Young (PDL 33) IMR90 cells were exposed to etoposide 20 µM for
24 h and then cultured for 11 days before harvesting. Cellular senescence was
verified by SA-β-gal staining. At least 300 cells were counted. Representative
images of control and etoposide-treated cells are reported. (c, d) Quantitative
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CDD-11-0226v2
Faraonio et al.
SUPPLEMENTARY INFORMATION
real-time PCR (Q-RT-PCR) analysis of cyclin A, thymidylate synthase (TYMS),
cyclin-selective ubiquitin carrier protein (UBE2C), interleukin-6 (IL6), and
forkhead box M1 (FOXM1) mRNAs in treated versus untreated IMR90 cells.
Results are the mean of triplicate determination.
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CDD-11-0226v2
Faraonio et al.
SUPPLEMENTARY INFORMATION
Supplementary Figure 3
Supplementary Figure 3  Effect of rapamycin on mTOR activity. IMR90
cells (PDL33) were electroporated with the indicated pre-miRNAs. After 24 h,
cells were harvested (day 0) or, where indicated, treated with 10 nM rapamycin
for 3 days. Lysates were analyzed for expression of ribosomal protein S6
phosphorylated at Ser240/244; -tubulin levels served as a loading control.
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CDD-11-0226v2
Faraonio et al.
SUPPLEMENTARY INFORMATION
Supplementary Figure 4
Supplementary Figure 4 A miRNA circuit sustaining cellular
senescence. Cellular senescence is associated to DNA damage and oxidative
stress that lead to an altered miRNA expression profile. In turn, de-regulated
miRNA may contribute to sustain DNA damage, oxidative stress and altered cell
cycle and, therefore, the execution of the senescence program. Some validated
targets of the senescence-associated miRNAs that might be involved in
mediating cell cycle arrest or potentiating the DNA damage (feed-forward loop)
are indicated. See text for details.
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CDD-11-0226v2
Faraonio et al.
SUPPLEMENTARY INFORMATION
Supplementary Methods
Western blotting. IMR90 cells were harvested following washing with PBS.
Cells were lysed by resuspension in a buffer containing 0.02 M HEPES (pH 7.9),
0.4 M NaCl, 0.1% NP-40, 10% (v/v) glycerol, 1 mM NaF, 1 mM sodium
orthovanadate, and a protease inhibitor cocktail (Sigma Chemical Co. St. Louis,
MO). Extracts were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide
gel electrophoresis, followed by blotting to nitrocellulose. The blots were probed
with antibodies to p21WAF1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA
catalog #SC 6246), p16INK4a (catalog #SC 65224), GAPDH (catalog #SC
69778), phospho-S6 Ribosomal protein (Ser240/244) (Cell Signaling, catalog
#2215) and -tubulin (Sigma Chemical, Co). Blots were developed by using
ECL kit (Amersham Biosciences, Uppsala, Sweden).
Real time PCR. For the quantitative analysis of mRNA levels in IMR90 cells,
SYBR Green-based Q-RT-PCR was used. Briefly, 1g of total RNA was
reverse transcribed by using random examers (pdN6) and Reverse
Transcriptase (M-MLV Reverse Transcriptase, Invitrogen, Carlsbad, CA).
Aliquots of the cDNA (1/10 of RT reactions) were amplified in triplicate using the
iQTM SYBR Green Supermix (Bio-Rad, Hercules, CA) in an iCycler iQ Real-Time
PCR Detection System (Bio-Rad). The following primers were used:
5’-TGGGCACTGCTGCTATGCT-3’ (cyclin A forward);
5’-TTTCTTGGTGTAGGTATCATCTGTAATGT-3’ (cyclin A reverse);
5’-GAGGAGTTGCTGTGGTTTATCAAG-3’ (thymidylate synthase, forward);
5’-CCCAGGCTGTCCAAAAAGTC-3’ (thymidylate synthase, reverse);
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CDD-11-0226v2
Faraonio et al.
SUPPLEMENTARY INFORMATION
5’-GGGATTTCTGCCTTCCCTGA-3’ (UBE2C forward);
5’-GCATTGTAAGGGTAGCCACTGG-3’ (UBE2C reverse);
5’-GTACATCCTCGACGGCATCTC-3’ (IL6, forward);
5’-GGTTCAGGTTGTTTTCTGCCA-3’ (IL6, reverse);
5’-AGCCCTTTGCGAGCAGAA-3’ (FOXM1, forward);
5’-CCACTGGATGTTGGATAGGCTAT-3’ (FOXM1, reverse);
5’-TGGAGATAACACTCTAAGCATAACTAAAGGT-3’ (ABL, forward)
5’-GATGTAGTTGCTTGGGACCCA-3’ (ABL, reverse).
c-ABL mRNA was used as endogenous control. Quantitative data analysis
followed the ΔΔCt method by the formula: 2-(sample 1 Ct - sample 2 Ct), where Ct is
the difference between the amplification fluorescent thresholds of the mRNA of
interest and the mRNA of c-ABL.
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