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Supplementary Methods and Figure Legends
Methods
Cell Cycle Analysis
Exponentially growing or plateau phase cells were collected following 24 hr PF05212384 exposure. To assess for cell cycle abrogation following treatment,
exponentially growing UMSCC1 cells were exposed to PF-05212384 for 24 hr,
irradiated, washed twice with 37°C drug free medium and subsequently collected as a
function of time following irradiation. At collection, cells were trypsinized, washed with
PBS, and fixed with ice-cold 70% ethanol in HBSS with subsequent refrigeration at 4°C.
Following >12 hr refrigeration, stored samples were washed with PBS, spun down at
1,000rpm for 5min, supernatant discarded, and the cell pellets suspended in .5-1mL of
0.1% Triton X-100 containing 500 ng/mL of DNase-free RNase for 30 minutes at 37°C,
followed by the addition of with 20 µg/mL of propidium iodide. Propidium iodide
stained cells were then immediately analyzed by a BD FACSCalibur (BD Biosciences)
flow cytometer and assessed for cell cycle distribution with CellQuest/MOD-Fit software
(VeritySoftware House).
Immunohistochemistry
OCT embedded tissue samples were cross-sectioned into 5-6 µm slices on glass slides (Histoserv,
Inc.) and stored at -70°C. Glass slides were then individually removed and air dried at room
temperature (RT) for 15 minutes. Tumor sections were fixed in ice-cold acetone for 15 min,
rinsed with PBS, permeabilized by incubation with 1% Triton X-100 in 1xPBS for 5 min, rinsed
with PBS, and blocked with non-serum protein blocker (DakoCytomation) at RT for 15 min.
Tissue sections were then incubated with primary antibody in PBS according to manufacturer
dilution recommendations with one drop of non-serum protein blocker per section at RT for 2 hr.
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Tissue sections were rinsed in PBS 3 x 5 min and subsequently incubated with the appropriate
Alexa Fluor secondary antibody (Abcam) at a dilution of 1:500 in PBS with one drop of nonserum protein blocker per section for 1 hr at RT. After 3x5 minute PBS rinses, slides were
mounted with ProLong Gold antifade with 4′,6-diamidino-2-phenylindole (Life Technologies).
Tissue sections were imaged with a Keyence BZ-9000 All-in-one Fluorescence Microscope
(Biorevo) at a 20x magnification. For fluorescence quantification, 3 fields per mouse tissue
section were chosen at random by DAPI nuclear staining; total fluorescence area normalized to
nuclear count per field was then determined. Total area for each group is subsequently expressed
relative to control average. Image analysis was performed with ImageJ (1). Total fluorescence
area mean and standard error of mean (SEM) were then calculated based on two or more
xenograft tissue sections. The following immunohistochemistry primary antibodies were used:
pS6 (Cell Signaling #5364) and Ki67 (Abcam ab15580).
RT-PCR Gene Expression Analysis
Exponentially growing UMSCC1 cells were plated (~100,000) in 100mm dishes and incubated at
37°C; 24 hr following initial plating the attached cells were irradiated (10 Gy). Immediately after
irradiation the exponentially growing cells were exposed to 10µM PF-05212384. Vehicle control
(10 µM DMSO) exposure and irradiation were conducted in parallel. At 24 hr post-irradiation,
samples for PCR array analysis were prepared serving as a 24 hr time point or cells were washed
twice with 37°C drug free medium and incubated at 37°C for an additional 48 hr then prepared.
Thus, the gene expression samples were collected at 24 and 72 hr post-irradiation time points.
The samples were prepared by first washing cells with ice cold 1xPBS then scraping them in the
presence of 2 ml TRIzol reagent (Life Technologies) per dish. TRIzol suspended cellular
contents were immediately snap frozen in liquid nitrogen. Liquid nitrogen frozen samples were
shipped to Qiagen (Frederick, Maryland) for senescence associated gene expression (84 total)
PCR array analysis (PCR Array Catalog #PAHS-050Z) via their RT2 Profiler PCR Array Service.
Briefly, Qiagen analyzed the provided samples utilizing a proprietary high-performance RT-PCR
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system in conjunction with dedicated PCR array data analysis software. RT-PCR analysis results
are expressed as fold-change versus control.
Irradiation
In vitro and in vivo experiments were irradiated with separate XRAD320 X-ray irradiators
(Precision X-ray, Inc., North Branford, CT) housed in the Radiation Biology Branch and Animal
Care Facility respectively of the National Cancer Institute. Unanesthetized mice were held in
custom plexiglass jigs shielded with lead leaving only the right tumor-bearing hind leg exposed to
ionizing radiation. For all experiments, ionizing radiation was delivered at a dose rate of ~2.42
Gy/min with 300kV X-rays at a distance of ~50cm from the radiation source.
Reference
1. Schneider CA, Rasband WS, Eliceiri KW. NIH image to ImageJ: 25 years of image analysis.
Nat Methods 2012;9(7):671-5.
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Supplementary Table S1
GENE
TP53
CDKN1A (p21)
CDKN1C (p57)
IRF5
GLB1 (-GAL)
ALDH1A3
FN1
(fibronectin)
CDKN2B
SERPINE1
IGFBP3
SPARC
THBS1
CCND1
CCNE1
CCNA2
CCNB1
CDK2
CDK4
CDK6
E2F1
EGR1
RB1
SOD2
CDKN1B (p27)
AKT1
*ATM
*GADD45A
*NBN
*PCNA
*TERF2
*TERT
*TP53BP1
DMSO Control + 10 Gy
24 hr
72 hr
1.7
1
3.4
2.1
1.6
1.6
1.4
0.7
1.2
0.81
1.4
0.94
PF-05212384†
24 hr
72 hr
15.6
1.2
4
1.8
9
1.4
3.4
1.6
1.8
0.79
1.5
3
PF-05212384† + 10 Gy
24 hr
72 hr
26.9
1.1
3.1
1.7
9.3
1.2
2.7
1.2
1.9
0.68
1.6
2.4
1.5
3
2.6
142.5
4.2
118.6
1.4
0.86
0.82
1
1
0.9
1.1
1
1.1
1.2
0.9
1.1
1.2
1.3
0.98
1.2
1
1.1
0.78
1.6
0.28
0.96
0.97
1.1
1.2
0.68
1.2
1.5
1.1
8
0.17
0.15
0.48
0.16
0.18
0.1
0.06
0.1
0.14
0.33
0.35
0.06
0.07
0.42
0.49
1.3
0.53
0.1
0.1
0.65
0.31
1.2
0.78
1.4
0.35
0.5
0.49
0.43
0.82
6.6
0.17
0.17
0.54
0.19
0.23
0.13
0.18
0.27
0.32
0.37
0.45
0.38
0.09
0.55
1.3
1.7
0.55
2.3
0.33
0.1
0.2
0.87
1.6
1.4
3.7
1.1
5.3
3.8
3.1
0.91
0.52
0.19
0.13
0.46
0.41
0.68
0.59
0.3
2.1
1.1
6
1.1
7.6
4.4
3
1.11
0.53
0.23
0.18
0.41
0.41
0.65
0.42
0.37
0.58
0.3
0.82
2.5
0.86
1.09
0.89
0.87
0.92
0.8
0.96
0.9
0.81
0.8
2.5
0.63
0.74
0.44
2.1
0.75
0.91
0.99
1.1
0.66
*DNA damage response gene, †10µM
0.41
0.48
0.3
0.75
0.5
0.56
1.3
0.27
0.53
0.49
0.51
0.68
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Supplementary Table S1. Senescence pathway directed RT-PCR genomic expression analysis
of UMSCC1 cells treated with 24 hour PF-05212384 (10 µM) or DMSO (10 µM) followed by
irradiation (10 Gy) or mock irradiation. Table representative of select genes from an 84 gene
array. Column time (24 hr and 72 hr) representative of time following irradiation. 72 hr time
point cells incubated with drug free medium at 37°C for 48 hr following drug removal. Values
expressed as fold change. Fold change (2
- ΔCt
) is the normalized gene expression (2
- ΔCt
) in the test
sample (10Gy, PF-05212384, PF-05212384+10Gy) divided by the normalized gene expression
-ΔCt
(2
) in the control sample. Genes with a relatively high (> 30) average threshold cycle,
meaning that its relative expression level was low in both control and test samples, were excluded
from analysis.
Supplementary Figure Legends
Supplementary Figure S1. Survival of UMSCC1 cells as a function of treatment time with PF05212384 (10µM) treatment alone, a function of time with PF-05212384 (10µM) treatment
followed by irradiation (8 Gy), or a function of time with DMSO control (10µM) treatment
followed by irradiation (8 Gy) (A). Survival as a function of PF-05212384 (10µM) treatment
time following irradiation (8 Gy) data not shown. Radiation survival curves for UMSCC1
plateau phase cells treated with PF-05212384 (10µM, Dashed Curve) or with DMSO Control
(10µM, Solid Curve) for 24 hours prior to irradiation (Pre-Treatment), (B), or 24 hours following
irradiation (Post-Treatment) (C). Column chart demonstrating a single representative experiment
of exponentially growing FaDu cells treated with PF-05212384 (1µM) or with DMSO Control
(1µM) for 24 hours prior to irradiation (Pre-Treatment) or 24 hours following irradiation (PostTreatment) (D). Individual survival curves representative of n = 2-3 independent experiments
fitted using the linear quadratic model. Dose modifying factors (DMFs) and drug treatment
toxicities are shown in Table 1.
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Supplementary Figure S2. A) Flow cytometry profiles of UMSCC1, UMSCC46, 1522, and
plateau-phase UMSCC1 cells treated with PF-05212384 (10 µM) for 24 hr with associated 24 hr
DMSO (10 µM) controls. B) Western analysis of the cell cycle proteins p27, p21, and p53 taken
from UMSCC1 and UMSCC46 following 24 hr PF-05212384 (10 µM) treatment. Western blots
representative of two independent experiments. Normalized protein densities for each band, with
Cytochrome C as the loading control, are in parenthesis below each row.
Supplementary Figure S3. The effects of PF-05212384 on the phosphorylation of the
PI3K/mTOR downstream targets S6 and 4EBP1 as a function of PF-05212384 (10 µM) treatment
time following irradiation (7.5 Gy) for exponentially growing A) UMSCC1 and B) UMSCC46
HNSCC cells.
Supplementary Figure S4. The effects of PF-05212384 (10µM) on UMSCC46 radiation
induced DNA damage repair. Western blot analysis of phosphorylated γH2AX from A)
UMSCC46 as a function of PF-05212384 (10µM) drug treatment time following irradiation (4
Gy) with associated B) mean (±SEM) densitometry of two independent γH2AX post-IR PF05212384 (PF-384) treatment westerns. Individual experiment densitometry results were
normalized based on the corresponding lane H2A loading control. Statistically significant
difference indicated by Ɵ as calculated by student’s t-test.
Supplementary Figure S5. Representative western blot of two independent experiments
characterizing the impact PF-05212384 has on ATM and DNA-PKc autophosphorylation and
total protein levels as a function PF-05212384 (10 µM) treatment time following irradiation (7.5
Gy) in exponentially growing UMSCC1 and UMSCC46 HNSCC cells (A). Western analysis of
total pDNA-PKc and DNA-PKc protein levels as a function of PF-05212384 (10 µM) treatment
time followed by irradiation (7.5 Gy) (B). Normalized protein densities for each band, with either
†
DNA-PKc or *HSC70 as a loading control, are in parenthesis below each row. S4(B) pDNA-
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PKc/DNA-PKc western analysis representative of multiple experiments, with repeat data at .5hr
affirming the above characteristic western blots.
Supplementary Figure S6. Heat map of individual mRNA absolute Ct obtained from
senescence pathway directed RT-PCR of UMSCC1 cells treated with 24 hour PF-05212384 (10
µM) or DMSO (10 µM) followed by irradiation (10 Gy) or mock irradiation. Column time (24 hr
and 72 hr) representative of time following irradiation. 72 hr time point cells incubated with drug
free medium at 37°C for 48 hr following drug removal. Green color representative of reduced
genomic expression beyond control, while red color representative of increased genomic
expression.