finally
performed
tunel
assays
facs
analysis
measuring
proportions
apoptotic
cells
investigate
whether
melk
involved
apoptosis
cascade
bcl-gl-related
pathway
tunel
assays
facs
analysis
revealed
overexpression
wild-type
melk
suppressed
bcl-gl-induced
apoptosis
d150a-melk
facs
analysis
cells
harvested
trypsinization
fixed
ethanol
room
temperature
minutes
centrifuging
remove
ethanol
cells
treated
pbs
containing
mg/ml
rnase
sigma-aldrich
minutes
followed
staining
pbs
containing
mg/ml
propidium
iodide
sigma-aldrich
minutes
cells
selected
from
least
ungated
cells
analyzed
dna
content
flow
cytometry
facs
calibur
becton
dickinson
san
diego
ca
usa
data
analyzed
using
cellquest
software
facs
calibur
becton
dickinson
assays
done
triplicate
independently
investigate
hypothesis
transiently
co-transfected
two
plasmid
clones
designed
express
ha-tagged
melk
wt
d150a
flag-tagged
bcl-gl
cos7
cells
performed
tunel
assay
facs
analysis
measure
proportions
apoptotic
cells
see
material
methods
shown
figure
5d
facs
analysis
cells
under
conditions
also
confirmed
overexpression
bcl-gl
increased
sub-g1
population
cells
compared
mock-transfected
cells
reported
previously
demonstrated
tunel
assay
facs
analysis
introduction
full-length
bcl-gl
cos7
cells
induced
apoptosis
however
under
conditions
addition
exogenous
wt-melk
suppressed
induction
apoptosis
bcl-gl
addition
d150a-melk
figure
5b
facs
analysis
cells
collected
transfection
pcaggsnhc
ha-mock
pcaggsn3fh
flag-mock
ha-tagged
melk
wt
d150a
flag-tagged
bcl-gl
expression
vectors
combinationsnotably
detect
cell-cycle-dependent
alteration
mdc1
s329/t331
phosphorylation
relative
total
mdc1
protein
content
using
flow
cytometry
suggests
sdtd
phosphorylation
occurs
throughout
interphase
supplementary
fig
s1b
onlinealtered
steady-state
levels
superoxide
shown
increased
oxidation
dhe
hct116
sirt3
overexpressing
cells
control
pcdna
vector
wild-type
sirt3
wt-sirt3
deacetylation
sirt3
mutant
mut-sirt3
cell
lines
analyzed
flow
cytometry
amount
hydrolyzed
dhe
per
cells
represented
mean
florescent
intensity
mfi