Supplementary Data
Supplementary Figure 1 – Scribble expression does not effect Myc driven apoptosis in
Emyc+ mice. (A) Following the development of Eµ-myc/scribble+/+ and Eµ-myc/scribble-/lymphomas in SCID mice, tumors were harvested and tissues were formalin fixed, paraffin
embedded and stained for TUNEL positive cells (Brown). Data are representative of independent
tumours harvested from 5 individual Scribble+/+Emyc+ and 4 individual Scribblenull/nullEmyc+.
Scale bar = 100M. Eµ-myc/scribble+/+ and Eµ-myc/scribble-/- lymphomas were incubated in vitro
for 24 hours with the indicated doses of etoposide. Cell viability was assessed by propidium iodide
staining (B) and TMRE (C). Data shown is the mean of three independent experiments ± SEM.
Tumours were isolated from 2 independent tumours per genotype. Data was determined to be not
significant by a student’s two-tailed t-test.
Supplementary Methods:
Etoposide: Pharma Pty Ltd, Mulgrave Australia
Propridium iodide assay
Eµ-myc/scribble+/+ and Eµ-myc/scribble-/- lymphomas were cultured at 5-8 x 105cells/ml in
24-well plates (CELLSTAR®, Greiner Bio-One, Kremsmünster, Austria) in Anne Kelso modified
DMEM (10% (v/v) heat-inactivated foetal calf serum (FCS), 0.1mM L-asparagine, 0.1mM
Glutamax,
2
mM
penicillin/streptomycin,
50uM
β-2-mercaptoethanol)
with
increasing
concentrations of etoposide (Pharma Pty Ltd, Mulgrave Australia) for 24 hours. After incubation,
200µl of cell suspension from each sample well was harvested into eppendorf tubes and centrifuged
at 1300rpm for 30seconds. The pellets were resuspended in 100µl PBS and transferred to FACS
tubes. 10µl of propidium iodide solution (Sigma, St Louis, MO, USA) was added from a diluted
propidium iodide stock (1mg/ml propidium iodide in PBS). Samples were analysed by flow
cytometry.
TMRE (tetramethylrhodamine ethyl ester) assay
Eµ-myc/scribble+/+ and Eµ-myc/scribble-/- lymphomas were set up at 5-8 x 105cells/ml. Cells were
cultured in 24-well plates (CELLSTAR®, Greiner Bio-One, Kremsmünster, Austria) in Anne Kelso
modified DMEM with increasing concentrations of etoposide for 24 hours. After incubation, 100µl
of cell suspension was harvested into FACS tubes and 100µl of TMRE (Invitrogen, Life
Technologies Corporation, Carlsbad, CA, USA) was added from a diluted stock (1/10,000 TMRE in
PBS). Samples were incubated at room temperature for 10-15 minutes before analysis by flow
cytometry.
TUNEL staining of tissue sections.
Immunohistological analysis of apoptotic cells from the lymph nodes of treated mice was
performed. Several sets of 4μM serial sections of formalin fixed, paraffin imbedded lymph nodes
were taken from random depths throughout the node. Two slides of each set were dewaxed and
subjected to TUNEL staining according to the protocol outlined in the ApopTag Peroxidase In Situ
apoptosis detection Kit (Cat #S7100, Millipore, Billerica, MA, USA.). Briefly, tissue sections were
deparaffinized and hydrated using a standard xylene/ethanol/PBS wash protocol. Tissue was then
treated with freshly diluted Proteinase K (20µg/mL) in PBS for 15 minutes at RT. Slides were
washed twice with water then endogenous peroxidase activity quenched with 3% hydrogen
peroxide for 5 minutes followed by two more washes in water. Equilibrium buffer (supplied by the
manufacturer) was then added directly to the tissue prior to incubation in a humidified chamber at
37°C for 1 hour with terminal deoxynucleotidyl transferase (TdT) enzyme. Following this
incubation, slides were then agitated for 15 seconds and then incubated for 10 minutes in
Stop/Wash buffer. Slides were then treated with anti-digoxignenin conjugate in the humidified
chamber at RT for 30 minutes. Slides were then washed thoroughly in PBS before peroxidase
substrate (Dako, Glostrup, Denmark) was added and developed for 4-6 minutes until optimal colour
development had occurred. Slides were then washed in three changes of dH2O and placed in Scotts
blue H2O for 30 seconds. Slides were dehydrated in three changes of xylene, 2 minutes each, and
then mounted under glass coverslips. Images were taken at 20x magnification on a BX51 Olympus
differential interference contrast microscope (Olympus, Center Valley, PA, USA).