Materials and Methods
Cell lines, transfections, plasmids and reagents
Cell lines used are: human osteosarcoma U20S cells; human A549 lung adenocarcinoma
cells; human small cell lung carcinoma H1299 cells; p53-/- and p73-/- mouse fibroblasts
[1,2]; and p53 null human osteosarcoma Saos-2 in which p73 expression can be induced
by addition of doxycycline [3].
p53-/- and p73-/- mouse embryonic fibroblasts were
spontaneously immortalized by the 3T3 passaging protocol [4] and used for experiments.
Transfections were carried out using RNAinfect reagent (Qiagen) for siRNA and
Lipofectamine 2000 (Invitrogen) for plasmids. p73 plasmids used have been described
previously [2]. Purvalanol A was purchased from Calbiochem and the following were
obtained from Sigma: MG132, cisplatin, roscovitine, cytochalasin D, nocodazole, taxol
and thymidine. RO-03066 was purchased from Alexis Biochemicals.
Cell-death assays
Cell viability was measured by several methods. Firstly, cell-death was measured by
propidium iodide (PI) exclusion assay. Treated (taxol, MG132, or other combinations as
described) cells were collected, including dead floating cells in the medium, washed 2X
in PBS before re-suspension in 2g/mL PI and % of dead cells up-taking PI measured by
flow cytometry (BD Biosciences FACScalibur). In addition, apoptosis was measured by
analysis of the sub-G1 DNA content, upon fixation of cells in 70% ethonal (as described
in cell cycle analysis). Moreover, apoptosis was determined by flow cytometry using
fluorescence labeled antibody recognizing cleaved caspase-3 (BD Biosciences).
1
Otherwise stated, all experiments were performed at least twice and average is presented.
The data are expressed as mean ± s.d. for triplicate.
Cell cycle, phospho-histone-H3 and MPM2 expression analysis
Samples were collected over indicated time points and fixed in 70% ethanol overnight.
For cell cycle analysis, fixed cells were treated with RNase for 20 minutes before
addition of 5g/mL PI and analyzed by FACS. For phospho-histone-H3 analysis, fixed
cells were washed 2X in PBS before incubating with 1:300 dilution of phospho-histoneH3 antibody (Upstate) for 1hr. After 2X wash with PBS, the samples are incubated with
secondary FITC-conjugated rabbit antibody for another hour. The samples are then
washed 3X in PBS before analysis with flow cytometry. For MPM2 expression analysis,
fixed cells were washed 2X in PBS before incubating with 1:100 dilution of FITC
conjugated MPM2 antibody (Abcam) for 1hr. The samples are then washed 2X in PBS
before flow cytometric analysis.
Immunofluorescence and Confocal microscopy
Cells were seeded onto glass coverslips (Marienfeld laboratory glassware) at 5x10 4
cells/ml in 6 well plates overnight and were treated with 200nM taxol for 18 hrs. For
mitochondria staining, 100nM mitotracker CMXRos (Molecular Probes Inc) were added
into culture medium for 15 minutes before fixing with 4% paraformaldehyde in PBS for
30 minutes at room temperature (RT). Fixed cells were washed with PBS and
permeabilized with 1% Triton X-100 in PBS for 10 minutes. After washing with PBS, the
coverslips were incubated with Bax antibody (N20, Santa Cruz Biotechnology Inc)
diluted in 5% BSA/PBS for 1 hr at RT. They were then washed twice with 0.02% Tween
2
20 and 1% BSA in PBS, followed by incubation with Alexa Flour 488 conjugated antirabbit (Molecular Probes Inc) for 30 minutes at RT. After washing 3X with 0.02% Tween
20 and 1% BSA in PBS, the coverslips were mounted with Vectorshield containing
DAPI (Vector Labs, CA). Similarly, for Bim and cleaved-caspase-3 co-staining,
coverslips were incubated with anti-Bim antibody (#2819, Cell Signaling) diluted in 5%
BSA/PBS for overnight at RT. They were then washed once with 0.02% Tween 20 and
1% BSA in PBS, followed by incubation with Alexa Flour 594 conjugated anti-rabbit
(Molecular Probes Inc) for 30 minutes at RT. The coverslips were then washed once
before incubation with Alexa Flour 488 conjugated anti-cleaved-caspase-3 antibody
(#9669, Cell Signaling) overnight. After washing 3X with 0.02% Tween 20 and 1% BSA
in PBS, the coverslips were mounted as described above. Cells were examined under a
Ziess LSM 510 laser scanning fluorescence confocal microscope.
Western blot analysis
Western blot analysis was performed as previously described [2]. For p73 detection,
200g of protein is loaded in each lane. The blotted membranes were immunostained
with antibodies specific for the following antigens: p73 (Clone ER-15, recognizing
TAp73a and TAp73b - Oncogene Science, Cambridge, MA), p53 (Novo Castra), Bubr1,
caspase-2 (BD Bioscience), PLK1, Mad2, Bax, cdc20, cdh1, p21, ANT, cytochrome C
(Santa Cruz Biotechnology Inc), Caspases-3,8,9, Bim, Puma, Bik, PARP (Cell Signaling
Technology Inc), Actin (Sigma), phospho-H3 (Upstate).
3
Cell fractionation
Cells were washed 2X with cold PBS, followed by centrifugation at 1500rpm for 5
minutes. Cell pellet was resuspended in fractionation buffer (22mmol/L mannitol, 70
mmol/L sucrose, 50 mmol/L PIPES-KOH, pH 7.4, 50mmol/L KCI, 5mmol/L EGTA,
2mmol/L MgCL2 and 1mmol/L dithiothreitol and protease inhibitors (Sigma). After 30
minutes incubation on ice, cells were homogenized by pulling through a 25G needle 20X.
The homogenate was spun at 8000rpm for 15 minutes. Supernatants containing
mitochondria fraction was removed and further spun at 13000rpm for 15 minutes. The
supernatant were removed and used as cytosol fraction. The pellet is washed once and
lyzed with 2% CHAPS in PBS for 15 minutes, spun at 13000rpm for 15 minutes and
lysate used as crude mitochondria extract.
siRNAs
siRNA were purchased from Qiagen. The target sequences are as follows - p73.1: AAA
AGC TGA TGA GGA CCA CTA, p73.2: AAG GCA ATA ATC TCT CGC AGT, p73.3:
AAC GGA TTC CAG CAT GGA CGT, p73.4: AAC TCT ACT GCC AGA TCG CCA,
p53: AAG ACT CCA GTG GTA ATC TAC, mad2: AAA CCT TTA CTC GAG TGC
AGA, bubr1: AAG GGA AGC CGA GCT ATT GAC, PLK1: AAC CAG TGG TTC
GAG AGA CAG, caspase-2: AAA CAG CTG TTG TTG AGC GAA, caspase-8: AAG
GAG CTG CTC TTC CGA ATT, caspase-9: AAG CTT CGT TTC TGC GAA CTA,
Bim: ACC GAG AAG GTA GAC AAT T, cdc20: AAC CTG GCG GTG ACC GCT
ATA, cdh1: AAT GAG AAG TCT CCC AGT CAG, negative control siRNA (cat no.
1022076, Qiagen).
4
Semi-quantitative PCR
1st strand cDNA is made using Superscript II (Invitrogen) and PCR was performed using
Taq polymerase (Qiagen) in a 30L solution according to manufacturers’ instructions
from 5g of total RNA. PCR reaction was amplified for 33 cycles at 94oC for 50s, 55oC
for 30s and 72oC for 30s for mouse Bim and p73. Gapdh and mouse p53 was amplified
for 20 cycles. Sequence of primers used for human p73 and gapdh have been described
previously [2]. Other primers used include – mouse p73-for: CAC TTC GAG GTC ACC
TTC CAG and mouse p73-rev: GCA CAA GCT CCA TCA GTT CTA G; mouse mdm2for: GGC TGT AAG TCA GCA AGA CTC and mouse mdm2-rev: CCA GGT AGC
TCA TCT GTG TTC; mouse p53-for: GGA ATT CGC CAT GGA GGA GTC ACA
GTCG and mouse p53-rev: CCC AAG CTT CAG TCT GAG TCA GGC CCC AC;
mouse Bim-for: AGG TGG ACA ATT GCA GCC TGC and mouse Bim-rev: TGA ACT
CGT CTC CGA TCC GCC; human mdm2-for: ATG TGC AAT ACC AAC ATG TCT
GTA CCT and human mdm2-rev: AGG GGA AAT AAG TTA GCA CAA TCA TTT
GA ; human Bim-for: AGC CCA GCA CCC ATG AGT TGT GAC and human Bim-rev:
CTC TGG GCG CAT ATC TGC AGG. 1st strand cDNA is made using Superscript II
(Invitrogen) according to manufacturers’ instructions
Chromatin Immunoprecipitation
2M doxocycline was added to Saos2-73 tetracylcine inducible cells. 12 hrs later, the
cells were fixed with 1% formaldehyde for 15 mins at room temperature (RT), and the
reaction was by addition of glycine to final concentration of 125 mM for 15 mins. Cells
were then washed twice with PBS and once with KM buffer (pH 6.8, 10mM NaCl,
1.5mM MgCl2, 1mM EGTA, 5mM dithiothreitol, 10% glycerol, 10mM MOPS, 10 mins
5
interval, and lysed for 30 mins using 4mL KM buffer-1% NP40 + protease inhibitors at
4oC. Thereafter, the buffer was adjusted with 2.7mL 5M NaCl for 60 mins at 4oC, cells
collected with TE buffer and the extracts were then sonicated 5 times with 10s pulse for 5
mins each, with intervals of about 10 mins on ice.
Samples were centrifuged at
13000rpm at 4oC for 30 mins and supernatant was collected. 400L of sample was used
for immunoprecipitation with 10l of p73 (Ab3, Oncogene Science, Cambridge, MA) or
HA antibody (Santa Cruz Biotechnology Inc) for 2 hrs at 4oC. 10l of Dynabeads
Protein A and G (Invitrogen) were added for a further 2 hrs. Immuno-complexes were
washed with 2x RIPA buffer, 1x HS buffer (0.1% SDS, 1% Triton X, 2mM EDTA,
20mM Tris-HCl, pH 8, 500mM NaCl), 1x LS buffer (0.1% SDS, 1% Triton X, 2mM
EDTA, 20mM Tris-HCl, pH 8, 150mM NaCl), 1x 0.25M LiCl buffer, 1x 0.5M LiCl
buffer at 37oC for 10 mins, follow by 2x RIPA buffer and 2x TE buffer. DNA protein
complex was eluted with TE buffer containing 1% SDS. Protein DNA complex was then
reverse crosslinked by addition of 200mM NaCl for 4 hrs at 65oC, followed by proteinase
K treatment at final concentration of 500g/mL at 55oC for 2 hrs. DNA was eluted using
DNA extraction kit (Qiagen) and used for PCR analysis. Primers used were as follows:
RE1-For: GAC AGC TGA GGT CTA GCT CAG CAC, RE1-Rev: ACG CTT TGT GTT
TAA CTG CAA TAC; RE2-For: CAG GGC TTA TGA GCC ATA GGG A, RE2-Rev:
CTT GTA TGC TAA TCT GCT GTG; RE3-For: CCC AAA GTA TCT TGA GGT TCC,
RE3-Rev: CTA TAT GCA ATT GGA GAC ATG GG; MDM2RE-For: GAT CGC AGG
TGC CTG TCG GGT CAC TA, MDM2RE-Rev: GGT CTA CCC TCC AAT CGC CAC
TGA ACA CA.
6
For analysis of p73 binding onto bim promoter at various stages of cell cycle, H1299 cells
were collected as follows  after starvation in serum free medium for 72hrs (for G0/G1);
arrested with 2mM thymidine for 24 hrs (G1/S); arrested with 2mM thymidine for 24 hrs
sand release in normal medium for 15 hrs (G2/M). H1299 cells also were treated with
100 nM of Taxol or 30 uM of cisplatin (CDDP) for 6 hrs after release from thymidine
block before harvesting for ChIP analysis. Intensity of bands were measured using
software AlphaEase FC (Alpha Imager 2200), and the ratio of specific/non-specific
binding was calculated by dividing the values obtained with anti-p73 antibody over that
of anti-HA antibody.
Statistical Analysis
Student's t test was used to test for statistical significance of the differences between the
different group parameters.
p values of less than 0.05 was considered statistically
significant.
7
References
1.
Lin KW, Nam SY, Toh WH, Dulloo I, Sabapathy K (2004) Multiple stress signals
induce p73beta accumulation. Neoplasia 6: 546-557.
2.
Toh WH, Kyo S, Sabapathy K (2005) Relief of p53-mediated telomerase
suppression by p73. J Biol Chem 280:17329-17338.
3.
Vikhanskaya F, Toh WH, Dulloo I, Wu Q, Boominathan L, Ng HH, Vousden K,
Sabapathy K (2007) p73 supports cellular growth through c-Jun-dependentAP-1
transactivation. Nat Cell Biol 9:698-705.
4.
Schreiber M, Kolbus A, Piu F, Szabowski A, Mohle-Steinlein U, Tian J, Karin M,
Angel P, Wagner EF (1999). Control of cell cycle progression by c-Jun is p53 dependent.
Genes Dev 13: 607-619.
8
Supplementary Figure Legends
Supplementary Figure 1. Taxol-induced cell death and MPM2 expression are reduced
upon treatment with PurvA, roscovitine or RO-3066
A.
Schematic shows protocol for treatment of cells with taxol in the presence or
absence of the various inhibitors (top panel). Thymidine arrested H1299 cells were
released into taxol and in the absence of presence of RO-3066 (10M), or were treated
with 20nM purvalanol A (PurvA), or 10M MG132, or 10M Roscovitine for 12-15 hrs
post-taxol treatment (without RO-3066)(when cells were in mitosis) and harvested 12 hrs
later. Cell viability was determined by % propidium iodide (PI) exclusion. The results
represent the mean + s.d. from at least three independent experiments (A). MPM2
analysis was performed by flow cytometric analysis. % MPM2 expression is indicated
(B).
Supplementary Figure 2. PurvA, roscovitine and RO-3066 treatment reduces number
of cells in mitosis upon taxol treatment and decreases cell death
H1299 cells were treated as indicated in the schematic in the top panel and as described
above, and cells were harvested and used for cell cycle profile analysis. In addition,
H1299 cells were treated with same concentration of PurvA, MG132, Roscovitine or RO3066 alone for 12 hrs and used for cell cycle profile analysis.
The % of sub-G1
population is indicated. Experiments for Taxol+Roscovitine were repeated with a
separate Taxol alone control (right panel).
9
Supplementary Figure 3.
Thymidine block does not affect cell death induced by
cisplatin
H1299 cells were blocked with thymidine and released into normal medium (top panel)
or 50M cisplatin (CDDP)-containing medium in the presence (middle) or absence
(lower panel) of thymidine, and cell death and cell cycle profile was analyzed after 24
hrs. % dead cells are indicated by the sub-G1 population.
Supplementary Figure 4.
Silencing p73 expression results in reduction in taxol-
mediated death in several cell systems
A-B.
H1299, A459 and U2OS cells were transfected with control and 4 different p73-
specific siRNAs individually and arrested at the G1/S phase of the cell cycle by treatment
with 2mM thymidine for 24 hrs, after which the medium was replaced with either normal
medium or 100nM taxol containing medium. Samples were collected 24 hrs post release
from thymidine block and used for RT-PCR analysis (A) or for assessment of caspase-3
cleavage (B). UT: untreated.
Supplementary Figure 5. Cell cycle analysis of taxol-treated mouse fibroblasts
Wild-type (WT), p53-/- and p73-/- mouse fibroblasts were treated with taxol and collected
at the indicated time-points for cell cycle analysis. 8N population indicates polyploid
cells.
10
Supplementary Figure 6. PLK silencing leads to G2/M arrest and cell death
Expression of PLK1 was silenced in H1299 cells that were arrested at the G1/S phase by
thymidine block for 24 hrs prior to release into the cell cycle. Control siRNA was used as
indicated. % dead cells (sub-G1 population) is indicated.
Supplementary Figure 7. MG132-induced cell death
Asynchronous (upper panel) and thymidine blocked (lower panel) H1299 cells were
treated with MG132 for 16 hrs, and cell viability was determined by propidium iodide
(PI) staining. For thymidine-blocked cells, MG132 was added upon release from
thymidine block. % dead cells are indicated. Duplicate experiments are shown for each
case.
Supplementary Figure 8. Taxol-induced death is reduced by dominant-negative forms
of p73
A.
% caspase-3 cleavage was determined using H1299 cells transfected with empty
vector (pcDNA), TAp73 or the dominant negative p73DD plasmids 24 hrs prior to taxol
treatment. Cell death was analyzed 24 hrs after taxol treatment, as described.
B.
Similar analysis was carried out using DNp73, the transactivation domain
deficient version of p73. Experiments were performed in duplicates and at least twice,
and mean + s.d. is shown.
11