Primary antibodies used were: 53BP1 (Bethyl Laboratories #A300-273A), BrdU (BD, 7580), CD31 (Invitrogen #HMCD3101), CD44 (Invitrogen #HMCD4401), CD45 (Invitrogen #HMCD4501), CD105 (Invitrogen #HMCD10520), Phospho-Chk1 (Ser345) (CST #2341), Phospho-Chk2 (Thr68) (CST #2661), CENP-A (Abcam #ab13939), human anti-centromeric autoantibody, α-CREST (Antibodies Inc., #15-235), γH2AX (Millipore #05-636). Secondary antibodies were AlexaFlour® conjugated donkey antibodies (Invitrogen).
Human ADSC isolation and expansion.
Human adipose derived stem cells were isolated from human subcutaneous white adipose tissue collected during liposuction procedures. The lipoaspirate was suspended in Hank's Buffered Salt Solution (HBSS), 3.5% Bovine Serum Albumin (BSA), 1% Collagenase, type II (Sigma) in 1:3 w/v ratio and shaken at 37°C for 50 min. The cells were filtered through a 70 µm mesh cell strainer (BD Falcon #352350), treated with Red Blood Cell Lysis buffer (150 mM NH4
Cl, 10 mM KHCO3
, 0.1 mM EDTA, pH 7.3), and expanded ex vivo in DMEM/F12 complete medium (DMEM/F12, 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B; Invitrogen) in 10% CO2
at 37°C and passaged at 80% confluency, changing medium every 72–96 h. Cumulative population doublings were calculated by summing the population doublings (PD = log(N/N0
) × 3.33
, where N0
is the number of cells plated in the flask and N is the number of cells harvested at this passage) across multiple passages as a function of the number of days it was grown in culture.
Surface marker characterization.
Five × 105 cells each were incubated for 30 min on ice in the dark with fluorochrome-conjugated antibodies (CD31, CD44, CD45 and CD105; Invitrogen) in PBS with 1% BSA (Sigma), washed and analyzed in a Guava EasyCyte Mini System (Guava Technologies, Millipore). Data analysis was done with FlowJo software (Tree Star, Ashland, OR).
Assessment of cellular senescence.
Senescence-associated β-galactosidase activity assay was done as described in manufacturer's kit (BioVision).
1–3 × 104 cells/well in 4-well slides were fixed with 4% paraformaldehyde and permeabilized with PBS, 0.5% Triton X-100. The blocking and antibody incubations were performed in 4% normal donkey serum (NDS; Jackson Immunochemicals) in PBS. The nuclei were counter-stained with 100 ng/ml 4′, 6-diamidino-2-phenylindole (DAPI; Sigma), and the slides were mounted in ProLong® Gold antifade aqueous mounting medium (Invitrogen). Analysis was performed as described in details in Supplemental Materials and Methods.
Human Cell Cycle qRCR Array (SABiosciences #PAHS-020C-2) was used for the profiling of self-renewing (SR) and senescent (SEN) hADSCs. Details of the experimental protocol and data analysis are given in Supplemental Materials and Methods online.
ChIP-Seq: nucleosomal ChIP and SoLiD library preparation.
Nucleosomal ChIP was performed as described in reference 31
, and in Supplemental Materials and Methods online. Samples were sequenced on SOLiD™ System 2.0 (Applied Biosystems) according to manufacturer's protocol.
5-Fluorouridine labeling for RNA transcript detection in situ.
Senescent hADSCs, 1 × 104 cells/well in 4-well slides, were treated with 2 mM 5-Fluorouridine (FUr; Sigma) for 10 min. The nuclei were exposed with ice-cold CSK buffer (100 mM KCl, 300 mM sucrose, 10 mM Pipes pH 6.8, 3 mM MgCl2, 1 mM EGTA, 1.2 mM phenylmethylsulfonyl fluoride) with 0.5% Triton X-100 and fixed with 4% paraformaldehyde. Immunostaining was performed as described above for immunoflourescence.
RT PCR analysis of retrotransposal transcription and qPCR.
Genomic coordinates of the Alu
elements tested in RT-PCR were from the March 2006 Assembly (NCBI36/hg18) of the Human Genome Browser at UCSC (http://genome.ucsc.edu
); MIR: chr10: 41922815–41922906, Alu: chr10:41928992–41929118, AluJb: chr21:10141132–10141429 and AluSx: chr21:10145344–10145644. 100 ng of total RNA was used with the RT2
First Strand Kit (SABiosciences) per reaction. The primers for first strand synthesis are at locations outside of the Alu
element sequences (external or reverse primers, Table S3
) and forward primers within the Alu
element sequence (internal forward primers, Table S3). RPL13A was used as a positive control.
Lentiviral shRNA constructs.
Lentiviral shRNA constructs to knockdown genetic Alu
transcript were designed as follows: oligonucleotides Lenti sh-132 Alu
RNA 5′-GAT CCC C
CC ACC ACG CCC GGC TAA TTT TCA AGA GA
A ATT AGC CGG GCG TGG TGG TTT TTG GAA A
-3′ and Lenti sh-193 Alu
RNA 5′-GAT CCC CC
C CGG GTT CAA GCG ATT CTT TCA AGA GA
A GAA TCG CTT GAA CCC GGG TTT TTG GAA A
-3′, were annealed with equal amounts of their complementary strands, creating restriction site specific overhangs for cloning, and ligated into HindIII and BglII digested, gel purified pENTR/pTER+
The constructs were confirmed by sequencing (sense strand sequence is shown above). Equal amounts of each constructs was mixed with pLenti-CMV-GFP DEST vector67
in LR Clonase reaction to recombine cloned shRNA production elements into a destination vector according to manufacturer's instructions (Invitrogen). The produced lentiviral plasmid was transformed into E. coli
Stbl3 cells (Invitrogen) for amplification.
Lentiviral production and transduction.
293T cells were grown in DMEM complete medium (DMEM, high glucose, 10% FBS, 0.1 M nonessential amino acid, 6 mM L-glutamine, 1 mM pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin) for lentiviral production and transfected for 12 h with pLenti sh-132Alu or pLenti sh-193Alu and pCgpV, pRSV-Rev and pCMVVSV-G helper plasmids (Allele Biotechnology) in 2:1:1:1 molar ratio using Lipofectamine 2000 (Invitrogen) according to standard protocol.67
Medium was collected at 48 and 72 h and filtered (0.45 µm). Virus was precipitated with PEG and frozen in aliquots (−80°C). Lentiviral transductions were done in complete medium with 5 µg/ml Polybrene (Santa Cruz Biotechnology) for 12 h. Viral titers were determined by comparing GFP positive cells counts to total population.
For each condition in 3[H]-thymidine uptake assay, 10,000 cells were treated with 1 µCi 3[H]-thymidine (Perkin-Elmer, Boston, MA) in DMEM/F12 complete for 24 h. DNA was isolated from harvested cells and quantified with NanoDrop (ND-1000; NanoDrop Technologies Inc.). 3[H]-thymidine uptake into cellular DNA was measured with liquid scintillation counter (LS 6500; Beckman Instruments). Lentiviral transduction was done as above, and untreated and polybrene treated controls were performed in parallel.
RNA was isolated from SR and SEN hADSCs (with or without lentiviral transduction) with mir-VANA (Ambion, Invitrogen) kit and 2 mg of total RNA/lane was run on a 7 M Urea, 6% polyacrylamide, TBE. Gel was stained with ethidium bromide, photographed and electroblotted onto Hybond™-N+ (Amersham, GE Healthcare). Hybridizations were performed in 6x SSC, 4x Denhardts', 0.1% SDS at 37°C. Oligonucleotide probes were labeled with Biotin-16-dUTP (Roche) by terminal transferase (NEB). Northern was visualized with streptavidin-HRP (Invitrogen) using ECL Plus western Blotting Detection Reagent (Amersham, GE Healthcare) and Amersham Hyperfilm (GE Healthcare). Oligonucleotide used as probes were:
Alu 132: 5′-CCA CCA CGC CCG GCT AAT TT-3′ and Alu 90: 5′-CGC GCG CCA CCA CGC CCG GCT AAT TTT TGT ATT TTT AGT AGA GAC GGG GTT TCA CCA TGT TGG CC-3′.