Sick Of All TheRucaparibChit Chat? I Am At This Website On Your Behalf

icalclusteringwasperformedinMatlabusingEuclideandistancemetricsandaveragelinkage.
4
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NATURECOMMUNICATIONS|DOI:10.1038/ncomms9390
ARTICLE
listofvalidatedantibodiesandabbreviations),signaltransduction Analysis of CycIFdata. meanwhile As a biologicallyrelevant testof CycIF
S235/6
V600E
(EGFR and p-S6
) and cell morphology (microtubules). In we imaged BRAF
melanoma cells (Fig. 3, Supplementary
otherexperimentswealsouseddyessuchasMitoTrackerRedto Fig. 9), in which the therapeutically bene?cial effects of Raf
labelmitochondriaandotherorganelles(SupplementaryFig.7).

inhibition are opposed by Daptomycin counter-therapeutic upregulation of
Thechoiceofwhichantibodiestocombineateachstepismade MAPK,Akt/mTORandothercompensatoryprogrowthpathways
27
empirically,withthegoalofassigningAlexa647totheweakest (so-called adaptive responses ). The level of ribosomal protein
signalssincethenear-infraredhaslessauto?uorescence.Relative S6 phosphorylation is the best-validated biochemical marker of
to?owcytometry(orCyTOF),imagingaddsinformationoncell growth inhibition by vemurafenib since S6 lies downstream of
22,28
morphology and protein translocation, which is particularly bothMAPKandAkt/mTORsignallingcascades
useful for studying the cytoskeleton, membrane receptor clus- phosphorylated sites pS6
.Thedoubly
S235/236
and pS6S240/244 are jointly
tering and nuclear foci. In the images shown in Fig.

2b and modi?ed by p70 S6-Kinase 1, which is regulated by TORC1 ;
S235/236
isalsophosphorylatedbythep90ribosomalS6kinase
29
SupplementaryFig.8,weperformedthreeroundsofCycIFand
pS6
morphometric features then extracted from ?ve channels using (RSK), which is regulated by the Raf/MEK/ERK cascade
26
30
Bayesianclassi?ersandsimilarmethods www.selleckchem.com/products/birinapant-tl32711.html .Thedimensionalityof (Fig. 3a) . Unsupervised clustering of single-cell eight-channel
such data is substantially higher than the number of unique CycIF data show that pS6S235/236 and pS6S240/244 levels are
antibodies or dyes and has the advantage, relative to ?ow strongly correlated at a single-cell level in dimethyl sulfoxide
cytometry,thatfeaturessuchasnuclearfociorreceptorclusters (DMSO)-treated cells (with Pearson��s correlation coef?cient
2
can be scored and correlated (Fig.

2c). For example, VEGFR2 R ?0.90)
but
less
so
following
exposure
to
implying
0.1mM
clusteringappearstobenegativelycorrelatedwiththenumberof
PCNAfoci,whichisamarkerofS-phase(Fig.2c,leftpanels).
vemurafenib
breakdown in the relationship between the S6 regulatory
(R2?0.53)
for
24h
(Fig.
3b)
a
a
b
DMSO control
Vemurafenib
p-H3
p21
p-S6
S235/236
PI3K-Akt
signaling
B-Raf
(V600E)
p-S6S240/244
Ki-67
p-Rb
MAPK
signaling
DNA
TORC1
p70S6K
2nd
DNA
1st
Vemurafenib (0.1 ?M)
RSK
p-Rb
p-S6S235/236
p21
p-S6S235/S236
p-S6S240/S244
Ki-67
p-S6S240/244
S6
p-H3
DNA
2nd
DNA1st
c
Density plot
Colored by p21 level (log AU)
Colored by Ki-67 level (log AU)
9
8
7
6
5
G1-S (68%)
G2 (21%)
9
8
7
6
10
9
8
7
6
5
10
9
8
7
6
9
8
7
6
G0 (10%)
5 14.6
15
15.4
14.6